Hypoxia-inducible factor-1 (HIF-1) is a heterodimeric transcription factor that governs cellular responses to reduced oxygen availability by mediating crucial homeostatic processes and is a major survival determinant for tumor cells growing in a low-oxygen environment. Clinically, HIF-1? seems to be important in pancreatic cancer, as HIF-1? correlates with metastatic status of the tumor. Extracellular superoxide dismutase (EcSOD) inhibits pancreatic cancer cell growth by scavenging nonmitochondrial superoxide. We hypothesized that EcSOD overexpression leads to changes in the O2(-)/H2O2 balance modulating the redox status affecting signal transduction pathways. Both transient and stable overexpression of EcSOD suppressed the hypoxic accumulation of HIF-1? in human pancreatic cancer cells. This suppression of HIF-1? had a strong inverse correlation with levels of EcSOD protein. Coexpression of the hydrogen peroxide-removing protein glutathione peroxidase did not prevent the EcSOD-induced suppression of HIF-1?, suggesting that the degradation of HIF-1? observed with high EcSOD overexpression is possibly due to a low steady-state level of superoxide. Hypoxic induction of vascular endothelial growth factor (VEGF) was also suppressed with increased EcSOD. Intratumoral injections of an adenoviral vector containing the EcSOD gene into preestablished pancreatic tumors suppressed both VEGF levels and tumor growth. These results demonstrate that the transcription factor HIF-1? and its important gene target VEGF can be modulated by the antioxidant enzyme EcSOD.
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.
Renewed interest in using pharmacological ascorbate (AscH-) to treat cancer has prompted interest in leveraging its cytotoxic mechanism of action. A central feature of AscH- action in cancer cells is its ability to act as an electron donor to O2 for generating H2O2. We hypothesized that catalytic manganoporphyrins (MnP) would increase AscH- oxidation rates, thereby increasing H2O2 fluxes and cytotoxicity. Three different MnPs were tested (MnTBAP, MnT2EPyP, and MnT4MPyP), exhibiting a range of physicochemical and thermodynamic properties. Of the MnPs tested, MnT4MPyP exerted the greatest effect on increasing the rate of AscH- oxidation as determined by the concentration of ascorbate radical [Asc•-] and the rate of oxygen consumption. At concentrations that had minimal effects alone, combining MnPs and AscH- synergized to decrease clonogenic survival in human pancreatic cancer cells. This cytotoxic effect was reversed by catalase, but not superoxide dismutase, consistent with a mechanism mediated by H2O2. MnPs increased steady-state concentrations of Asc•- upon ex vivo addition to whole blood obtained either from mice infused with AscH- or patients treated with pharmacologic AscH-. Finally, tumor growth in vivo was inhibited more effectively by combining MnT4MPyP with AscH-. We concluded that MnPs increase the rate of oxidation of AscH- to leverage H2O2 flux and ascorbate-induced cytotoxicity.
Using a GWA analysis of a comprehensive glioma specimen population, we identified whole gain of chromosome 19 as one of the major chromosomal aberrations that correlates to patients outcomes. Our analysis of significant loci revealed for the first time NOTCH3 as one of the most significant amplification. NOTCH3 amplification is associated with worse outcome compared to tumors with non-amplified locus. NOTCH receptors (NOTCH1-4) are key positive regulators of cell-cell interactions, angiogenesis, cell adhesion and stem cell niche development which have been shown to play critical roles in several human cancers. Our objective is to determine the molecular roles of NOTCH3 in glioma pathogenesis and aggressiveness. Here we show for the first time that NOTCH3 plays a major role in glioma cell proliferation, cell migration, invasion and apoptosis. Therefore, our study uncovers the prognostic value and the oncogenic function of NOTCH3 in gliomagenesis and supports NOTCH3 as a promising target of therapy in high grade glioma. Our studies allowed the identification of a subset of population that may benefit from GSI- or anti-NOTCH3- based therapies. This may lead to the design of novel strategies to improve therapeutic outcome of patients with glioma by establishing medical and scientific basis for personalized chemotherapies.
Poor prognosis and resistance to therapy in malignant gliomas is mainly due to the highly dispersive nature of glioma cells. This dispersive characteristic results from genetic alterations in key regulators of cell migration and diffusion. A better understanding of these regulatory signals holds promise to improve overall survival and response to therapy. Using mapping arrays to screen for genomic alterations in gliomas, we recently identified alterations of the protein tyrosine phosphatase receptor type kappa gene (PTPRK) that correlate to patient outcomes. These PTPRK alterations are very relevant to glioma biology as PTPRK can directly sense cell-cell contact and is a dephosphorylation regulator of tyrosine phosphorylation signaling, which is a major driving force behind tumor development and progression. Subsequent sequencing of the full length PTPRK transcripts revealed novel PTPRK gene deletion and missense mutations in numerous glioma biopsies. PTPRK mutations were cloned and expressed in PTPRK-null malignant glioma cells. The effect of these mutations on PTPRK anti-oncogenic function and their association with response to anti-glioma therapeutics, such as temozolomide and tyrosine kinase inhibitors, was subsequently analyzed using in vitro cell-based assays. These genetic variations altered PTPRK activity and its post-translational processing. Reconstitution of wild-type PTPRK in malignant glioma cell lines suppressed cell growth and migration by inhibiting EGFR and ?-catenin signaling and improved the effect of conventional therapies for glioma. However, PTPRK mutations abrogated tumor suppressive effects of wild-type PTPRK and altered sensitivity of glioma cells to chemotherapy.
Redox regulation of epidermal growth factor receptor (EGFR) signaling helps protect cells against oxidative stress. In this study, we investigated whether the cytotoxicity of an EGFR tyrosine kinase inhibitor, erlotinib (ERL), was mediated by induction of oxidative stress in human head and neck cancer (HNSCC) cells. ERL elicited cytotoxicity in vitro and in vivo while increasing a panel of oxidative stress parameters which were all reversible by the antioxidant N-acetyl cysteine. Knockdown of EGFR by using siRNA similarly increased these oxidative stress parameters. Overexpression of mitochondrial targeted catalase but not superoxide dismutase reversed ERL-induced cytotoxicity. Consistent with a general role for NADPH oxidase (NOX) enzymes in ERL-induced oxidative stress, ERL-induced cytotoxicity was reversed by diphenylene iodonium, a NOX complex inhibitor. ERL reduced the expression of NOX1, NOX2, and NOX5 but induced the expression of NOX4. Knockdown of NOX4 by using siRNA protected HNSCC cells from ERL-induced cytotoxicity and oxidative stress. Our findings support the concept that ERL-induced cytotoxicity is based on a specific mechanism of oxidative stress mediated by hydrogen peroxide production through NOX4 signaling.
Brain metastasis (BM) can affect ? 25% of nonsmall 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.
O?-methylguanine DNA-methyltransferase (MGMT) promoter methylation has been identified as a potential prognostic marker for glioblastoma patients. The relationship between the exact site of promoter methylation and its effect on gene silencing, and the patients subsequent response to therapy, is still being defined. The aim of this study was to comprehensively characterize cytosine-guanine (CpG) dinucleotide methylation across the entire MGMT promoter and to correlate individual CpG site methylation patterns to mRNA expression, protein expression, and progression-free survival. To best identify the specific MGMT promoter region most predictive of gene silencing and response to therapy, we determined the methylation status of all 97 CpG sites in the MGMT promoter in tumor samples from 70 GBM patients using quantitative bisulfite sequencing. We next identified the CpG site specific and regional methylation patterns most predictive of gene silencing and improved progression-free survival. Using this data, we propose a new classification scheme utilizing methylation data from across the entire promoter and show that an analysis based on this approach, which we call 3R classification, is predictive of progression-free survival (HR ?=?5.23, 95% CI [2.089-13.097], p<0.0001). To adapt this approach to the clinical setting, we used a methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) test based on the 3R classification and show that this test is both feasible in the clinical setting and predictive of progression free survival (HR ?=?3.076, 95% CI [1.301-7.27], p?=?0.007). We discuss the potential advantages of a test based on this promoter-wide analysis and compare it to the commonly used methylation-specific PCR test. Further prospective validation of these two methods in a large independent patient cohort will be needed to confirm the added value of promoter wide analysis of MGMT methylation in the clinical setting.
There is widespread agreement that cancer gene discovery requires high-quality tumor samples. However, whether primary tumors or cultured samples are superior for cancer genomics has been a longstanding subject of debate. This debate has recently become more important because federally funded cancer genomics has been centralized under The Cancer Genome Atlas, which has chosen to focus exclusively on primary tumors. Here, we provide a data-driven "perspective" on the effect of sample type selection on cancer genomics research. We show that, in the case of glioblastoma multiforme, primary tumors and xenografts are best for the identification of amplifications, whereas xenografts and cell lines are superior for the identification of homozygous deletions. We also note that many of the most important oncogenes and tumor suppressor genes have been discovered through the use of cell lines and xenografts, and highlight the lack of published evidence supporting the dogma that ex vivo culture generates artifactual genetic lesions. Based on this analysis, we suggest that cancer genomics projects such as The Cancer Genome Atlas should include a variety of sample types such as xenografts and cell lines in their integrated genomic analysis of cancer.
Malignant gliomas are the most frequent type of primary brain tumors. Patients outcome has not improved despite new therapeutics, thus underscoring the need for a better understanding of their genetics and a fresh approach to treatment. The lack of reproducibility in the classification of many gliomas presents an opportunity where genomics may be paramount for accurate diagnosis and therefore best for therapeutic decisions. The aim of this work is to identify large and focal copy number abnormalities (CNA) and loss of heterozygosity (LOH) events in a malignant glioma population. We hypothesized that these explorations will allow discovery of genetic markers that may improve diagnosis and predict outcome. DNA from glioma specimens were subjected to CNA and LOH analyses. Our studies revealed more than 4000 CNA and several LOH loci. Losses of chromosomes 1p and/or 19q, 10, 13, 14, and 22 and gains of 7, 19, and 20 were found. Several of these alterations correlated significantly with histology and grade. Further, LOH was detected at numerous chromosomes. Interestingly, several of these loci harbor genes with potential or reported tumor suppressor properties. These novel genetic signatures may lead to critical insights into diagnosis, classification, prognosis, and design of individualized therapies.
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