Curcumin is a polyphenolic compound derived from the Indian spice turmeric. We used nanoparticle-encapsulated curcumin to treat medulloblastoma and glioblastoma cells. This formulation caused a dose-dependent decrease in growth of multiple brain tumor cell cultures, including the embryonal tumor derived lines DAOY and D283Med, and the glioblastoma neurosphere lines HSR-GBM1 and JHH-GBM14. The reductions in viable cell mass observed were associated with a combination of G(2)/M arrest and apoptotic induction. Curcumin also significantly decreased anchorage-independent clonogenic growth and reduced the CD133-positive stem-like population. Down-regulation of the insulin-like growth factor pathway in DAOY medulloblastoma cells was observed, providing one possible mechanism for the changes. Levels of STAT3 were also attenuated. Hedgehog signaling was blocked in DAOY cells but Notch signaling was not inhibited. Our data suggest that curcumin nanoparticles can inhibit malignant brain tumor growth through the modulation of cell proliferation, survival and stem cell phenotype.

Hypoxic regions are frequent in glioblastoma (GBM), the most common type of malignant adult brain tumor, and increased levels of tumor hypoxia have been associated with worse clinical outcomes. To unmask genes important in hypoxia, we treated GBM neurospheres in hypoxia and identified monocarboxylate transporter-4 (MCT4) as one of the most upregulated genes. To investigate the clinical importance of MCT4 in GBM, we examined clinical outcomes and found that MCT4 overexpression is associated with shorter patient survival. Consistent with this, MCT4 upregulation correlated with the aggressive mesenchymal subset of GBM, and MCT4 downregulation correlated with the less aggressive G-CIMP (Glioma CpG Methylator Phenotype) subset of GBM. Immunohistochemical analysis of tissue microarrays confirmed that MCT4 protein levels were increased in high-grade as compared with lower-grade astrocytomas, further suggesting that MCT4 is a clinically relevant target. To test the requirement for MCT4 in vitro, we transduced neurospheres with lentiviruses encoding short-hairpin RNAs (shRNAs) against MCT4, resulting in growth inhibition of 50–80% under hypoxia in two lines. MCT4 knockdown was associated with a decreased percentage of cells expressing the stem-cell marker CD133 and increased apoptotic fraction. We also found that flow-sorted CD133-positive cells had almost sixfold higher MCT4 levels than CD133-negative cells, suggesting that the stem-like population might have a greater requirement for MCT4. Most importantly, MCT4 silencing also slowed GBM intracranial xenograft growth in vivo. Interestingly, whereas MCT4 is a well-characterized lactate exporter, we found that both intracellular and extracellular lactate levels did not change following MCT4 silencing, suggesting a novel lactate export-independent mechanism for growth inhibition in GBMs. To identify this potential mechanism, we performed microarray analysis on control and shMCT4-expressing neurospheres and found a dramatic reduction in the expression of multiple Hypoxia-Inducible Factor (HIF)-regulated genes following MCT4 knockdown. The overall reduction in HIF transcriptional response was further validated using a hypoxia response element (HRE)-dependent green-fluorescent protein (GFP) reporter line.

Chromosomal instability (CIN), a high rate of chromosome loss or gain, is often associated with poor prognosis and drug resistance in cancers. Aneuploid, including near-polyploid, cells contain an abnormal number of chromosomes and exhibit CIN. The post-mitotic cell fates following generation of different degrees of chromosome mis-segregation and aneuploidy are unclear. Here we used aneuploidy inducers, nocodazole and reversine, to create different levels of aneuploidy. A higher extent of aneuploid and near-polyploid cells in a given population led to senescence. This was in contrast to cells with relatively lower levels of abnormal ploidy that continued to proliferate. Our findings revealed that senescence was accompanied by DNA damage and robust p53 activation. These senescent cells acquired the senescence-associated secretory phenotype (SASP). Depletion of p53 reduced the number of senescent cells with concomitant increase in cells undergoing DNA replication. Characterisation of these SASP factors demonstrated that they conferred paracrine pro-tumourigenic effects such as invasion, migration and angiogenesis both in vitro and in vivo. Finally, a correlation between increased aneuploidy and senescence was observed at the invasive front in breast carcinomas. Our findings demonstrate functional non-equivalence of discernable aneuploidies on tumourigenesis and suggest a cell non-autonomous mechanism by which aneuploidy-induced senescent cells and SASP can affect the tumour microenvironment to promote tumour progression.

The most commonly utilized class of chemotherapeutic agents administered as a first-line therapy are antimitotic drugs; however, their clinical success is often impeded by chemoresistance and disease relapse. Hence, a better understanding of the cellular pathways underlying escape from cell death is critical. Mitotic slippage describes the cellular process where cells exit antimitotic drug-enforced mitotic arrest and "slip" into interphase without proper chromosome segregation and cytokinesis. The current report explores the cell fate consequence following mitotic slippage and assesses a major outcome following treatment with many chemotherapies, therapy-induced senescence. It was found that cells postslippage entered senescence and could impart the senescence-associated secretory phenotype (SASP). SASP factor production elicited paracrine protumorigenic effects, such as migration, invasion, and vascularization. Both senescence and SASP factor development were found to be dependent on autophagy. Autophagy induction during mitotic slippage involved the autophagy activator AMPK and endoplasmic reticulum stress response protein PERK. Pharmacologic inhibition of autophagy or silencing of autophagy-related ATG5 led to a bypass of G arrest senescence, reduced SASP-associated paracrine tumorigenic effects, and increased DNA damage after S-phase entry with a concomitant increase in apoptosis. Consistent with this, the autophagy inhibitor chloroquine and microtubule-stabilizing drug paclitaxel synergistically inhibited tumor growth in mice. Sensitivity to this combinatorial treatment was dependent on p53 status, an important factor to consider before treatment. Clinical regimens targeting senescence and SASP could provide a potential effective combinatorial strategy with antimitotic drugs. .

During normal development, heterogeneous expression of Notch ligands can result in pathway suppression in the signal-sending cell, a process known as lateral inhibition. It is unclear if an analogous phenomenon occurs in malignant cells. We observed significant induction of Notch ligands in glioblastoma neurospheres and pancreatic carcinoma cells cultured in low oxygen, suggesting that this phenomenon could occur around hypoxic regions. To model lateral inhibition in these tumors, the ligand Jagged1 was overexpressed in glioblastoma and pancreatic carcinoma cells, resulting in overall induction of pathway targets. However, when ligand high and ligand low cells from a single line were co-cultured and then separated, we noted suppression of Notch pathway targets in the former and induction in the latter, suggesting that neoplastic lateral inhibition can occur. We also found that repression of Notch pathway targets in signal-sending cells may occur through the activity of a Notch ligand intracellular domain, which translocates into the nucleus. Understanding how this neoplastic lateral inhibition process functions in cancer cells may be important in targeting ligand driven Notch signaling in solid tumors.

Glioblastomas (GBM) are the most common adult malignant brain tumors and contain a hypoxic/necrotic core surrounded by proliferative cells. To unmask genes important in hypoxia, we exposed 2 GBM neurosphere lines, HSR-GBM1 and JHH-GBM10, to 1% and 21% oxygen levels for 24 hours and compared gene expression using Agilent oligonucleotide microarrays. We identified SLC16A3 (Monocarboxylate transporter-4, MCT4) as one of the most upregulated genes in response to hypoxia. To investigate the clinical importance of MCT4 in GBM, we examined the Kaplan-Meier survival curves of glioma patients using public databases. We found that patients with upregulation of MCT4 (≥2.0X) have a significantly shorter survival (p<0.0001) than patients with intermediate expression. Consistent with this, MCT4 upregulation correlated with the aggressive mesenchymal subset of GBM (p<0.0001). We next examined MCT4 protein levels using immunohistochemical analysis of tissue microarrays, confirming that MCT4 protein levels were increased in high-grade as compared to lower grade astrocytomas (p<0.0001). These data clearly demonstrated that MCT4 is a clinically relevant target. We next tested the requirement of MCT4 in vitro. We found that when neurospheres were transduced with viruses encoding short hairpin RNA (shRNA) against MCT4, cell growth was inhibited by 30-69% in hypoxia. Interestingly, similar results were observed in normoxia, suggesting that MCT4 may be critical to tumor growth and survival independent of oxygen levels. We next explored the role that MCT4 may play in the maintenance of the stem-like population in our neurosphere lines. We found that MCT4 is overexpressed in sorted CD133-positive cells compared to CD133-negative cells in both HSR-GBM1 and JHH-GBM10. Furthermore, flow cytometric analysis of shMCT4-expressing neurospheres showed a decrease in the percentage of CD133-positive cells as compared to controls. This suggested to us that MCT4 silencing might inhibit stem-like cell proliferation/survival, offering a potential explanation for growth inhibition following MCT4 knockdown in normoxia. To test this hypothesis, we examined the effect of MCT4 inhibition functionally using a clonogenic assay. Cells were exposed to normoxia or hypoxia for 48 hours followed by recovery in normoxia. We found that MCT4 silencing resulted in significant reduction in sphere number and size in both normoxic and hypoxic cells, consistent with a loss of clonogenicity. In agreement with what we found in vitro, MCT4 silencing also slowed GBM intracranial xenograft growth in vivo (p=0.009). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3483. doi:1538-7445.AM2012-3483

We attempted to treat medulloblastoma and glioblastoma, malignant tumors of the central nervous system, with curcumin, a polyphenolic compound derived from the Indian spice turmeric. Curcumin was delivered in a nanoparticle-encapsulated formulation (nanocurcumin) to increase its solubility and bioavailability. Nanocurcumin caused a dose-dependent decrease in cell growth as measured by MTT in multiple brain cancer cell lines, including the embryonal tumor derived cultures DAOY, D283, and PFSK, and the glioblastoma neurosphere line HSR-GBM1. Notably, doses used in these studies did not cause a comparable inhibition in the growth of NIH 3T3 cells or non-neoplastic human fetal cortical neurospheres. The reductions in viable cell mass observed were associated with a combination of G2/M arrest and apoptotic induction. The proportion of G2/M cells increased between 25% and 106% in the various lines, while the percentage of apoptotic cells increased at least two-fold. Nanocurcumin was also found to reduce the CD133+ stem-like cancer cell population in medulloblastoma and glioblastoma cells. In the primary glioblastoma culture JHH-GBM14, the CD133+ population decreased from 7.7% to 0.6%. In addition, 10uM nanocurcumin suppressed clonogenicity of our brain tumor cell lines by more than 97%. Curcumin has been shown to target multiple pathways in different tumor types. We found that Stat3 activity was reduced by nanocurcumin in the DAOY medulloblastoma cell line, via reductions in phospho-Tyr705 and phospho-Ser727. Nanocurcumin also reduced the expression of Hes 5, one of the target genes in the Notch pathway. However, we did not find significant changes in protein expression of Bcl2, MEK, ERK and Akt following nanocurcumin treatment. In conclusion, our results suggest that nanocurcumin can inhibit malignant brain tumor growth, at least in part due to reduction in Stat activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4440.

Chromosomal rearrangements resulting in novel fusion genes are among the most prevalent form of genetic alterations known in cancer, and numerous examples exist in both adult and childhood malignancies. To date, however, none have been reported in pediatric high-grade glioma (pHGG), so we have undertaken to search for novel structural rearrangements using three distinct techniques. Firstly, we took a candidate approach and screened a series of 83 pHGG for the fusion previously described in adult glioblastoma between PDGFRA and KDR (VEGFR2) at 4q12. Using RT-PCR and sequencing, we identified the second reported instance of KDR:PDGFRA in a single case of glioblastoma (age 1.2 years). Next, we applied the iCNA algorithm to identify copy number aberrations with intragenic breakpoints using Affymetrix 500K SNP data from a cohort of 100 pHGG, identifying two candidates arising from genomic amplification and intrachromosomal rearrangement in an analogous mechanism to KDR:PDGFRA. We finemapped the breakpoints using custom Agilent oligonucleotide arrays and characterized the fusions DHX57:MAP4K3 (2p22) and CSGALNACT2:RET (10q11) in cases of anaplastic astrocytoma (2 years) and recurrent glioblastoma (12.8 years), respectively. Finally, we sequenced the entire genomes of five pediatric glioma cell lines at .30× coverage using the Illumina HiSeq2000 platform, and screened for rearrangements using the BreakDancer (BD) package. We identified a median of 165 intragenic structural variants per genome that were filtered based on BD confidence score, number and orientation of reads and by visual inspection using IGV software. Candidate fusions being systematically validated and screened in our pHGG cohorts include interchromosomal rearrangements resulting in TULP4:RPTOR (t6;17 -SF188), GORASP2:CDADC1 (t2;13 -KNS42) and C15ORF57:CBX3 (t15;7 -UW479). These data highlight the presence of hitherto unrecognized fusion genes in pHGG which may play important roles in the unique biology of the tumors as well as provide excellent candidates for novel therapeutic strategies. Recently, we and others identified the first recurrent somatic mutations in a histone gene (H3F3A) in one-third of pediatric glioblastoma (pedGBM), the most devastating brain tumor in childhood. The observed mutations in H3F3A, coding for the non-canonical histone variant H3.3, led to amino acid substitutions at two critical residues of the histone tail (K27M and G34R), at or near sites of important post-translational modifications. Furthermore, we have shown that pedGBMs carrying K27M or G34R mutations are characterized by distinct gene expression and DNA methylation profiles. This clearly shows that different signalling pathways predominate in K27M or G34R mutated tumors. To address the functional impact of these different H3.3 mutations in more detail, we generated GBM cell lines stably expressing either K27M or G34R mutated H3.3. K27M mutated cells show a reduction of trimethylated H3K27 (H3K27me3) as indicated by western blot. Using immunohistochemistry on a tiss...