Kah Jing Lim

A polymeric nanoparticle formulation of curcumin inhibits growth, clonogenicity and stem-like fraction in malignant brain tumors

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 G2/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. See commentary: Indian gold treating cancer in the age of nano

Inhibition of monocarboxylate transporter-4 depletes stem-like glioblastoma cells and inhibits HIF transcriptional response in a lactate-independent manner

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.

Hypoxia Promotes Uveal Melanoma Invasion through Enhanced Notch and MAPK Activation

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.

Abstract 3776: Hypoxia-inducible factor 1α (HIF1α) has a prometastatic effect in uveal melanoma .

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC 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<0.0001) and greatly impaired cellular invasion both in normoxia and in hypoxia (p<0.0001), suggesting that HIF1α is necessary for invasion even under normoxic conditions. In order to determine the downstream targets responsible for such profound effect of HIF1α on cellular invasion, we examined expression of Notch pathway members, which we have previously linked to uveal melanoma invasion. Interestingly, hypoxia induced mRNA expression of the Notch ligand Jag2 and target Hey1 by up to 3 fold. We also analyzed the expression of Snail, which is known to promote invasion and metastasis of tumors outside the eye, and which can be regulated by HIF1α. We observed more than 2 fold increase of Snail mRNA levels under hypoxic conditions in OCM1 cells, and this induction was abolished by suppressing HIF1α using shRNA. These data suggest that HIF1α plays a crucial role in promoting metastatic spread in uveal melanoma cells, possibly by regulating Notch activity or expression of the Snail transcription factor. 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

Abstract 2930: Silencing MCT4 inhibits GBM growth, HIF response, and CD133-positive fraction in a lactate-independent fashion.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC 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<0.0001). Using immunohistochemical analysis, we also found that MCT4 protein levels are increased in high-grade as compared to lower grade astrocytomas (p<0.0001), confirming the clinical importance of MCT4. To test the requirement for MCT4 under hypoxia in vitro, we silenced MCT4 in neurospheres using lentiviruses encoding short hairpin RNA (shRNA) specific for MCT4. Cell growth was inhibited in hypoxia by ∼60% in HSR-GBM1 and ∼70% in JHH-GBM10 in these neurosphere lines following MCT4 knockdown. Interestingly, while MCT4 was expressed at lower levels in normoxia, silencing it in 21% oxygen also significantly inhibited growth. CD133-positive stem-like cells expressed a significantly higher amount of MCT4 compared to CD133-negative cells, and decreased proliferation in vitro following MCT4 silencing was associated with reduced CD133-positive stem-like cells and increased apoptosis. This suggests that MCT4 is critical for the survival of CD133-positive stem-like cells under hypoxia, and its inhibition targets the stem cell pool in the hypoxic niche in tumors. Importantly, MCT4 silencing also slowed GBM intracranial xenograft growth in vivo (p=0.009). Interestingly, while multiple earlier studies had identified and characterized MCT4 as a lactate exporter, we found that both extracellular and intracellular lactate levels did not change following MCT4 silencing. Instead, MCT4 inhibition led to downregulation of the HIF (Hypoxia-Inducible Factor) response, reducing HIF target gene expression and pointing to a potential novel lactate-independent function of MCT4 and a unique mechanism for growth inhibition in malignant gliomas. 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

Abstract 5049: Targeting the Notch and mTOR pathways in diffuse intrinsic pontine glioma.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC 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<0.005) in treated DIPG cells compared to non-treated cells and a dose-dependent reduction in phospho-S6, with concomitant upregulation of phospho-AKT 473 indicating TORC2 activation. Dual TORC1 and TORC2 inhibition by combining a rapalog and a gamma-secretase inhibitor may prove to be synergistic in DIPG. Simultaneous treatment with a rapalog and gamma-secretase inhibition suppresses TORC1 and TORC2, as measured by western blot. We are currently investigating the possible mechanism of action of these drugs in DIPG. Our laboratory is investigating additional metabolic inhibitors that can be used with TORC1 and TORC2 inhibitors to further improve treatment options for DIPG. We are currently exploring the role of the Notch and mTOR pathways in DIPG in pre-clinical studies in mice, with the goal of moving targeted inhibitors into clinical trials for these currently highly lethal tumors. 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

Abstract 3483: Inhibition of Monocarboxylate Transporter 4 (MCT4) targets stem-like cells in glioblastoma

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL 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

Abstract 3335: The potential role of lateral inhibition in the maintenance of glioblastoma cancer stem cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL 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

Abstract 2480: High-level activation of the Notch pathway in diffuse intrinsic pontine glioma

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, Children9s 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

Abstract 4440: Using nanocurcumin to treat medulloblastoma and glioblastoma

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC 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.