Kah Suan Lim

BRAF Activation Induces Transformation and Then Senescence in Human Neural Stem Cells: A Pilocytic Astrocytoma Model

Purpose: BRAF is frequently activated by gene fusion or point mutation in pilocytic astrocytoma, the most common pediatric brain tumor. We investigated the functional effect of constitutive BRAF activation in normal human neural stem and progenitor cells to determine its role in tumor induction in the brain. Experimental Design: The constitutively active BRAFV600E allele was introduced into human neurospheres, and its effects on MAPK (mitogen-activated protein kinase) signaling, proliferation, soft agarose colony formation, stem cell phenotype, and induction of cellular senescence were assayed. Immunohistochemistry was used to examine p16INK4a levels in pilocytic astrocytoma. Results: BRAFV600E expression initially strongly promoted colony formation but did not lead to significantly increased proliferation. BRAFV600E-expressing cells subsequently stopped proliferating and induced markers of oncogene-induced senescence including acidic β-galactosidase, PAI-1, and p16INK4a whereas controls did not. Onset of senescence was associated with decreased expression of neural stem cell markers including SOX2. Primary pilocytic astrocytoma cultures also showed induction of acidic β-galactosidase activity. Immunohistochemical examination of 66 pilocytic astrocytomas revealed p16INK4a immunoreactivity in the majority of cases, but patients with tumors negative for p16INK4a had significantly shorter overall survival. Conclusions: BRAF activation in human neural stem and progenitor cells initially promotes clonogenic growth in soft agarose, suggesting partial cellular transformation, but oncogene-induced senescence subsequently limits proliferation. Induction of senescence by BRAF may help explain the low-grade pathobiology of pilocytic astrocytoma, whereas worse clinical outcomes associated with tumors lacking p16INK4a expression could reflect failure to induce senescence or an escape from oncogene-induced senescence. Clin Cancer Res; 17(11); 3590–9. ©2011 AACR.

Pathological and molecular advances in pediatric low-grade astrocytoma.

Pediatric low-grade astrocytomas are the most common brain tumors in children. They can have similar microscopic and clinical features, making accurate diagnosis difficult. For patients whose tumors are in locations that do not permit full resection, or those with an intrinsically aggressive biology, more effective therapies are required. Until recently, little was known about the molecular changes that drive the initiation and growth of pilocytic and other low-grade astrocytomas beyond the association of a minority of cases, primarily in the optic nerve, with neurofibromatosis type 1. Over the past several years, a wide range of studies have implicated the BRAF oncogene and other members of this signaling cascade in the pathobiology of pediatric low-grade astrocytoma. In this review, we attempt to summarize this rapidly developing field and discuss the potential for translating our growing molecular knowledge into improved diagnostic and prognostic biomarkers and new targeted therapies.

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 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 3459: Expression of oncogene-induced senescence markers in pilocytic astrocytomaIs associated with younger age and longer survival

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Pilocytic astrocytomas are the most common pediatric brain tumor. They are low grade neoplasms, and in some cases can spontaneously stop growing or regress. The recent discovery of constitutive BRAF activation in the majority of pilocytic astrocytomas, due either to gene duplication/fusion or point mutation, suggests a possible molecular basis for this relatively benign pathobiology. Oncogenic BRAF-induced senescence has previously been identified in benign melanocytic nevi and colonic polyps, but whether it also occurs in pilocytic astrocytomas is not clear. To investigate this, we examined the senescence marker acidic beta-galactosidase in low passage pilocytic astrocytoma cultures established from freshly resected tumors. Two of these cultures, tested at passages 2 to 3, were derived from tumors that contained KIAA1549:BRAF fusions, and both expressed the senescence marker in over half of the cells. Another culture tested at passage 3 was also strongly positive for acidic beta-galactosidase, but the genetic status of its parent tumor is not known. A final culture came from a pilocytic astrocytoma lacking the KIAA1549:BRAF fusion, or an activating V600E BRAF point mutation, and was negative for the senescence marker, but was tested at passage 10. To help confirm these effects in vivo, we immunostained a tissue microarray containing 78 pilocytic astrocytoma for p16 and p53, both of which have been implicated in oncogene-induced senescence in other tumor types. The majority of pilocytic astrocytoma expressed p16, but the 13% of tumors lacking this senescence marker were associated with significantly shorter overall survival (p = 0.0002). Patients whose pilocytic astrocytomas lacked p16 were also older (mean age 25 years) as compared to those with intermediate (mean age 12 years) or high expressing tumors (mean age 7 years). Immunohistochemical analysis of p53 revealed much weaker staining, and only 18 tumors were positive, but expression correlated positively with that of p16 (Spearman r = 0.45, p = 0.0001). Our data suggest that the low-grade phenotype of pilocytic astrocytomas may be due to oncogene-induced senescence driven by KIAA1549:BRAF fusions or BRAF V600E point mutations, and that expression of senescence markers in vivo is associated with younger age at presentation and better clinical outcomes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3459. doi:10.1158/1538-7445.AM2011-3459

Abstract 3303: BRAF activation induces cellular transformation and senescence and down-regulates SOX2 in human neural stem cells: a model of pilocytic astrocytoma

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL In pilocytic astrocytomas, the primary molecular alteration is BRAF activation occurring via gene duplication/fusion or V600E point mutation. To investigate the mechanisms by which BRAF activation leads to transformation, BRAF V600E was introduced into cortical and cerebellar human fetal neural stem and progenitor cells. Overexpression of BRAF V600E did not lead to increases in cellular proliferation compared to controls, but did promote colony forming ability in soft agar. However, after initially growing well, BRAF V600E expressing cells subsequently showed decreased proliferation as compared to control cells, though they remained viable visually. Because constitutive BRAF activation in melanocytes usually leads to cellular senescence, with only rare lesions progressing to melanoma, we examined if senescent markers were induced in our system. Overexpression of BRAF V600E led to an increase in markers of oncogene-induced senescence, such as acidic beta-galactosidase and p16. FISH analysis revealed no differences in telomere length in BRAF over-expressing cells compared to GFP-transduced control cells, indicating that this phenotype was not due to telomere shortening. The pattern of initial transformation followed by senescence is similar to the frequently indolent course of pilocytic astrocytoma, suggesting that overexpressing constitutively active BRAF in human neural stem and progenitor cells phenocopies the behavior of this tumor. To further investigate the mechanism of oncogene-induced senescence, we examined SOX2 expression. This human somatic cell reprogramming factor is an important neurodevelopmental gene and is known to prevent the induction of cellular senescence. However, the functional role of SOX2 in gliomas is poorly understood. We found that although SOX2 was expressed in control GFP transduced neurospheres, high-level expression of BRAFV600E led to downregulation of SOX2 in association with senescence. Our data support the concept that BRAF oncogene-induced senescence plays a role in the pathobiology of pilocytic astrocytoma, and suggest that modulation of SOX2 may play an important role in this process. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3303. doi:10.1158/1538-7445.AM2011-3303