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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.

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.

The transcriptional response promoted by hypoxia-inducible factors has been associated with metastatic spread of uveal melanoma. We found expression of hypoxia-inducible factor 1α (HIF-1α) protein in well-vascularized tumor regions as well as in four cell lines grown in normoxia, thus this pathway may be important even in well-oxygenated uveal melanoma cells. HIF-1α protein accumulation in normoxia was inhibited by rapamycin. As expected, hypoxia (1% pO2) further induced HIF-1α protein levels along with its target genes VEGF and LOX. Growth in hypoxia significantly increased cellular invasion of all 5 uveal melanoma lines tested, as did the introduction of an oxygen-insensitive HIF-1α mutant into Mel285 cells with low HIF-1α baseline levels. In contrast, HIF-1α knockdown using shRNA significantly decreased growth in hypoxia, and reduced by more than 50% tumor invasion in four lines with high HIF-1α baseline levels. Pharmacologic blockade of HIF-1α protein expression using digoxin dramatically suppressed cellular invasion both in normoxia and in hypoxia. We found that Notch pathway components, including Jag1-2 ligands, Hes1-Hey1 targets and the intracellular domain of Notch1, were increased in hypoxia, as well as the phosphorylation levels of Erk1-2 and Akt. Pharmacologic and genetic inhibition of Notch largely blocked the hypoxic induction of invasion as did the pharmacologic suppression of Erk1-2 activity. In addition, the increase in Erk1-2 and Akt phosphorylation by hypoxia was partially reduced by inhibiting Notch signaling. Our findings support the functional importance of HIF-1α signaling in promoting the invasive capacity of uveal melanoma cells in both hypoxia and normoxia, and suggest that pharmacologically targeting HIF-1α pathway directly or through blockade of Notch or Erk1-2 pathways can slow tumor spread.

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.

Diffuse intrinsic pontine glioma (DIPG) is a universally fatal pediatric brain tumor which is known to have frequent alteration of mTOR signaling. Analysis of DIPG tissue obtained through the Mid-Atlantic DIPG Consortium (MADC) also shows high level activation of Notch pathway effectors HES4 and HES5. We have established JHH DIPG1, a human DIPG xenograft and neurosphere cell line from a rapid autopsy specimen obtained through the MADC. Notch 1 and 3 and Notch downstream effectors, Hes1, Hes5 and Hey1, are highly expressed in JHH DIPG1 and a second DIPG line, SU DIPG1. These DIPG cell lines also express high level phospho-S6 and phospho-AKT 473, indicating activation of TORC1 and TORC2. Notch and mTOR pathway signaling is equal to or exceeding that observed in glioblastoma neurosphere cell lines. Notch targeting using a gamma-secretase inhibitor leads to a dose-dependent reduction in cell proliferation of up to 30% (p=0.05) as well as inhibition of phospho-AKT 473, suggesting that inhibition of the NOTCH pathway targets TORC2 complexes. BrdU incorporation also showed that treatment of DIPG cell lines with a TORC1 inhibitor also has up to a 60% attenuation of cell proliferation (p Citation Format: Marianne Hutt, Wendy Goldstein, Javad Nazarian, Antoinette Price, Kah Jing Lim, Katherine Warren, Howard Chang, Charles Eberhart, Eric Raabe. Targeting the Notch and mTOR pathways in diffuse intrinsic pontine glioma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5049. doi:10.1158/1538-7445.AM2013-5049

Glioblastomas (GBM) contain a hypoxic core surrounded by proliferative cells. Our group and others have shown that GBM stem cells favor a hypoxic microenvironment, and it is believed that many reside in the tumor core. Conventional therapies target the tumor bulk, but may spare stem cells in the hypoxic niche. To patch this therapeutic loophole, we sought to target the GBM stem cell microenvironment by identifying genes that are important for survival in hypoxia. We identified monocarboxylate transporter 4 (MCT4) as one of the most upregulated genes in our GBM neurosphere lines in response to hypoxia. Clinically, GBM patients with a two-fold or more upregulation of MCT4 have a significantly shorter survival (p = 0.036) than patients with intermediate expression. Consistent with this data, MCT4 upregulation correlated with the aggressive mesenchymal subset of GBM (p Citation Format: Kah Suan Lim, Kah Jing Lim, Brent A. Orr, Antoinette C. Price, Charles G. Eberhart, Eli E. Bar. Silencing MCT4 inhibits GBM growth, HIF response, and CD133-positive fraction in a lactate-independent fashion. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2930. doi:10.1158/1538-7445.AM2013-2930

Melanomas arising from the uveal tract of the eye represent the most frequent primary intraocular malignancy in adults and are the second most common type of melanoma. Uveal melanomas are prone to disseminate hematogenously to the liver, and have an overall survival rate of approximately 50%. Hypoxia-inducible factor 1α (HIF1α), a master transcriptional regulator of the hypoxic response, is strongly associated with aggressive uveal melanomas. Elevated expression of one of the HIF target gene lysyl oxidase (LOX) has been found at the invasive front of primary tumors, and is associated with shorter metastasis-free survival. Here we investigated the mechanism responsible for the prometastatic effect of HIF1α in uveal melanoma. Western blot examination of HIF1α and HIF2α proteins in uveal melanoma cell lines grown in normoxia (21% pO2) and in hypoxia (1% pO2) revealed HIF2α to be low. In contrast, HIF1α protein was relatively abundant in the OCM1, OCM3, OMM1, Mel290 and 92.1 uveal melanoma lines grown in normoxia, and was further induced by up to 5 fold in hypoxia. The mRNA levels of VEGF and LOX, two transcriptional targets of HIF1α, were also induced by 2 to 4 fold in hypoxia. Interestingly, while hypoxia reduced the growth of these lines as measured by MTS assay, low oxygen tension increased the ability of uveal melanoma cells to invade Matrigel by 2 to 5 fold. Genetic suppression of HIF1α expression through shRNA in OCM1 and 92.1 cells significantly decreased cellular growth in hypoxia (p Citation Format: Laura Asnaghi, Michael H. Lin, Kah Suan Lim, Kah Jing Lim, Eli E. Bar, Charles G. Eberhart. Hypoxia-inducible factor 1α (HIF1α) has a prometastatic effect in uveal melanoma . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3776. doi:10.1158/1538-7445.AM2013-3776

Glioblastoma (GBM) is a malignant cancer of the central nervous system. Tumor cells are highly resistant to conventional therapies and tumors always recur. Cancer stem cells are thought to be critical for long-term growth and therapeutic resistance of GBM. It is therefore important to understand the signaling mechanisms maintaining GBM cancer stem cells. Notch is known to be an important signaling pathway during neural development, and can promote the maintenance of stem and progenitor cells via the process of lateral inhibition. Lateral inhibition is a contact-dependent signaling mechanism by which some cells within a field remain stem-like while others differentiate. In normal development, Notch ligands initiate this process by binding to the receptor on an adjacent cell, signaling to this neighbor to remain undifferentiated. Although Notch is known to be a critical player in the maintenance of stem-like cancer cells in various tumor types, including GBM, it is not known if the mechanism of lateral inhibition is also conserved in neural cancer stem cells. To test this hypothesis, we engineered a model system where isogenic cells expressing low and high levels of the Jag1 ligand are co-cultured followed by flow-based separation and determination of Notch pathway activity in each respective population. These experiments suggest that lateral inhibition is conserved in GBM. In the GBM neurosphere lines HSR-GBM1 and 040821, co-culture induced expression of Notch targets Hes5 and Hey1 in Jag1-low “signal receiving” cells and inhibited expression of these targets in Jag1-overexpressing “signal sending” isogenic cells. Preliminary experiments suggest that the ligand intracellular domain (ICD) is involved in this process. Our data suggest conservation of a developmental mechanism regulating Notch activation in cancer, and may affect our understanding of how to treat Notch-dependent tumors. 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 3335. doi:1538-7445.AM2012-3335

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 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

Diffuse intrinsic pontine glioma (DIPG) is a universally fatal pediatric brain tumor. Due to its critical location within the brainstem, and its characteristic MRI signature, biopsy is rarely performed. Since many patients die at home in hospice care, autopsy specimens have traditionally been rare. Because of this difficulty in obtaining tissue, three institutions (Johns Hopkins, Children's National Medical Center, and the National Institutes of Health - Pediatric Oncology Branch) have formed the Mid-Atlantic DIPG Consortium (MADC), to collect and disseminate rapid-autopsy material from patients with DIPG. Using a fresh tissue specimen obtained from a rapid autopsy and disseminated through the MADC, we have established a human DIPG xenograft and neurosphere cell line, JHH-DIPG1. In vitro, these cells are GFAP positive and express human neural stem cell markers often observed in glioblastoma, including human specific SOX2 and NESTIN. They also show marked invasive properties in vitro as measured by transwell invasion assay. In vivo, JHH-DIPG1 forms tumor xenografts in approximately six months in immunocompromised mice. Tumor cells diffusely infiltrate distant brain structures, recapitulating the invasive phenotype of DIPG. Due to their high level expression of neural stem cell markers, and the well-known roles of the Notch pathway in stem cell biology and aggressive brain tumors, we examined the expression of members of the Notch pathway in DIPG. Notch downstream effectors, specifically Hes1, Hes5 and Hey1, are highly expressed in JHH-DIPG1, at levels equal to or exceeding those observed in neurosphere cell lines derived from adult supratentorial glioblastoma. The JHH-DIPG1 tumor cell line represents one of only a handful of pediatric DIPG cell lines extant, and the high level of Notch pathway activation in these cells suggests that blockade of the Notch pathway using gamma secretase inhibitors and shRNAs against the canonical Notch effector CBF may be efficacious in DIPG. Our laboratory is actively investigating the role of the Notch pathway in DIPG using this and other DIPG cell lines, with the goal of moving Notch inhibition into clinical trials for these currently highly lethal tumors. 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 2480. doi:1538-7445.AM2012-2480