Abhinav Dey

Sonic Hedgehog Signaling Drives Mitochondrial Fragmentation by Suppressing Mitofusins in Cerebellar Granule Neuron Precursors and Medulloblastoma

Sonic hedgehog (Shh) signaling is closely coupled with bioenergetics of medulloblastoma, the most common malignant pediatric brain tumor. Shh-associated medulloblastoma arises from cerebellar granule neuron precursors (CGNP), a neural progenitor whose developmental expansion requires signaling by Shh, a ligand secreted by the neighboring Purkinje neurons. Previous observations show that Shh signaling inhibits fatty acid oxidation although driving increased fatty acid synthesis. Proliferating CGNPs and mouse Shh medulloblastomas feature high levels of glycolytic enzymes in vivo and in vitro. Because both of these metabolic processes are closely linked to mitochondrial bioenergetics, the role of Shh signaling in mitochondrial biogenesis was investigated. This report uncovers a surprising decrease in mitochondrial membrane potential (MMP) and overall ATP production in CGNPs exposed to Shh, consistent with increased glycolysis resulting in high intracellular acidity, leading to mitochondrial fragmentation. Ultrastructural examination of mitochondria revealed a spherical shape in Shh-treated cells, in contrast to the elongated appearance in vehicle-treated postmitotic cells. Expression of mitofusin 1 and 2 was reduced in these cells, although their ectopic expression restored the MMP to the nonproliferating state and the morphology to a fused, interconnected state. Mouse Shh medulloblastoma cells featured drastically impaired mitochondrial morphology, restoration of which by ectopic mitofusin expression was also associated with a decrease in the expression of Cyclin D2 protein, a marker for proliferation. Implications: This report exposes a novel role for Shh in regulating mitochondrial dynamics and rescue of the metabolic profile of tumor cells to that of nontransformed, nonproliferating cells and represents a potential avenue for development of medulloblastoma therapeutics. Mol Cancer Res; 14(1); 114–24. ©2015 AACR.

YB-1 is elevated in medulloblastoma and drives proliferation in Sonic hedgehog - dependent cerebellar granule neuron progenitor cells and medulloblastoma cells

Postnatal proliferation of cerebellar granule neuron precursors (CGNPs), proposed cells of origin for the SHH-associated subgroup of medulloblastoma, is driven by Sonic hedgehog (Shh) and insulin-like growth factor (IGF) in the developing cerebellum. Shh induces the oncogene Yes-associated protein (YAP), which drives IGF2 expression in CGNPs and mouse Shh-associated medulloblastomas. To determine how IGF2 expression is regulated downstream of YAP, we carried out an unbiased screen for transcriptional regulators bound to IGF2 promoters. We report that Y-box binding protein-1 (YB-1), an onco-protein regulating transcription and translation, binds to IGF2 promoter P3. We observed that YB-1 is upregulated across human medulloblastoma subclasses as well as in other varieties of pediatric brain tumors. Utilizing the cerebellar progenitor model for the Shh subgroup of medulloblastoma in mice, we show for the first time that YB-1 is induced by Shh in CGNPs. Its expression is YAP-dependent and it is required for IGF2 expression in CGNPs. Finally, both gain-of function and loss-of-function experiments reveal that YB-1 activity is required for sustaining CGNP and medulloblastoma cell (MBC) proliferation. Collectively, our findings describe a novel role for YB-1 in driving proliferation in the developing cerebellum and MBCs and they identify the SHH:YAP:YB1:IGF2 axis as a powerful target for therapeutic intervention in medulloblastomas.

Abstract B12: Yes-Associated Protein: A master metabolic regulator In medulloblastoma

Downstream of mitogenic Sonic hedgehog (Shh) signaling, Yes-Associated Protein (YAP) can drive proliferation in Cerebellar Granule Neural Progenitor (CGNP) cells. CGNPs are the proposed cells of origin of SHH medulloblastomas. They are a neural progenitor type whose developmental expansion requires signaling by Shh, a secreted ligand produced by the neighboring Purkinje neurons. Approximately 30% of human medulloblastomas bear an activated Sonic hedgehog pathway gene expression signature. Ectopic expression of YAP promotes highly aggressive Shh-driven medulloblastoma growth and radio-resistance (Fernandez et al., 2009). Medulloblastoma being the most common solid malignancy of childhood and a leading cause of pediatric mortality, the current standard of care results in about 60% “cure” rate. But the survivors are beset with long-term side effects, including cognitive impairment, seizures, premature aging, and susceptibility to cancer. Moreover, recurrence and metastasis are lethal. Therefore, identification of novel modes of molecular targeted therapies is critical for the improved quality of life for survivors and reduced incidence of recurrence and metastasis. Recently, there has been renewed interest in how altered metabolic patterns in tumors could be exploited for therapeutic purposes. Deregulating the metabolic machinery for aberrant energy utilization is one of the hallmarks of a proliferating cancer cell. Previously, our lab made the novel observation that Shh mitogenic/oncogenic signaling is tightly coupled to the reprogramming of mitochondrial bioenergetics: Shh inhibits fatty acid oxidation (FAO, or β-oxidation) while driving increased fatty acid synthesis (FAS), an early step of lipogenesis. The production of citrate, an essential component for fatty acid synthesis, occurs inside the mitochondrion via the Tri-carboxylic acid cycle (TCA). We analyzed the effect of Shh treatment and ectopic YAP expression on CGNPs and found that YAP increases levels of fatty acid synthase (FASN) and ATP citrate lyase (ACLY), while YAP knock-down in Shh-treated CGNPs resulted in reduced levels of these enzymes. Moreover, we also observed a surprising decrease in mitochondrial membrane potential. This prompted us to further analyze the ultrastructure of mitochondria using Transmission Electron Microscopy. Shh-treated or ectopic YAP-expressing mitochondria presented a swollen morphology, along with an expanded matrix space and deformed cristae structure, typical of morphologically aberrant mitochondria. These differences in mitochondrial structure were also visible in ultrastructures of SmoA1 tumor tissue as well as in vitro cultures of SmoA1 tumor cells (MBCs). Being dynamic structures, mitochondria undergo constant fusion and fission events, which contribute to their biogenesis. Expression of fusion genes Mitofusin 1 and 2 was reduced while DRP1, a fission promoting gene was highly induced in all samples under study. Ectopic expression of Mitofusin 1 and 2, and knock down of DRP1 in CGNPs and MBCs not only restores the membrane potential to the non-proliferating state, but also indicates a reduction in proliferation. Our study thus implicates YAP-regulated metabolic pathways and enzymes as potential targets for novel medulloblastoma therapies. Our goal is to determine whether hampering YAP-mediated mitochondrial fragmentation can restore the metabolic profile of tumor cells to that of non-transformed, non-proliferating cells, thus suggesting a potential novel treatment paradigm that may reduce or eliminate the requirement for high dose radiation. Citation Format: Anshu Malhotra, Abhinav Dey, Anna Marie Kenney. Yes-Associated Protein: A master metabolic regulator In medulloblastoma. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr B12.

Abstract 3196: STAT3 inhibitor WP1066 as a novel therapeutic for medulloblastoma

The purpose of this study is to determine the efficacy of the STAT3 inhibitor WP1066 on the sonic-hedgehog (SHH) subtype of medulloblastoma (MB). STAT3, or signal transducer and activator of transcription 3, is a potential novel target for the treatment of SHH-MB. We have generated substantial preliminary data showing that STAT3 activation promotes the survival of SHH-MB cancer stem cells (CSCs) that are critical for treatment resistance and tumor recurrence. Smoothened (SMO) antagonists have shown efficacy in treating subtypes of SHH-MB, however all tumors develop resistance and SMO inhibitors are ineffective for patients with SHH-MB harboring mutations downstream of SMO. Our preliminary in vitro data suggests that combined SMO and STAT3 inhibition has synergistic activity, and that constitutively activated STAT3 is protective against SHH-MB cell death mediated by SMO inhibition. Methods: The effect of increasing concentrations of WP1066 on Shh-responsive MB cells was assessed by Western blot analysis for STAT3 phosphorylation, WST assay for cell proliferation, and soft agar colony formation. The effect of WP1066 on apoptosis and the cell cycle was assessed by flow cytometry. An ex vivo murine SmoA1 SHH-MB brain slice culture method was used to assess the effects of WP1066 alone and in combination with radiation. In vivo studies consist of mice treated with 1) WP1066, 2) SMO inhibitor LDE225 3) WP1066 in combination with LDE225 and 4) vehicle alone. All mice are imaged by MRI prior to treatment and every 3 weeks after treatment. Tumor volume is calculated and rate of tumor growth and survival are measured. The mice are followed until death or the end of the experiment at 36 weeks. Results: In human Shh-responsive MB cells in vitro, WP1066 blocked IL6-induced STAT3 activation (phosphorylation), decreased cell proliferation and abrogated tumor colony formation. Results regarding the mechanism of inhibition of cell proliferation/viability by WP1066 (i.e. apoptosis and cell cycle analysis) are pending. In the SHH-MB brain tumor slice cultures, WP1066 treatment induced potent tumor cell kill, especially of the CSCs within the perivascular niche. WP1066 also showed synergy with radiation, but did not add toxicity to normal brain cells. Preliminary in vivo data indicate a statistically significant decrease in the rate of tumor growth and prolonged survival in mice treated with WP1066 compared to vehicle control. Results of the efficacy of WP1066 with LDE225 are pending. Conclusions: WP1066 has potent efficacy against Shh-responsive MB cells in vitro and SHH-MB tumors ex vivo, without toxicity to normal brain cells. Preliminary data is promising for WP1066 activity against SHH-MB in vivo, suggesting WP1066 is a novel therapeutic for SHH-MB. Citation Format: Laura K. Metrock, Jingbo Liu, Liangping Yuan, Hongying Zhang, Abhinav Dey, Tobey MacDonald. STAT3 inhibitor WP1066 as a novel therapeutic for medulloblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3196.

Abstract 2451: Cancer stem cell survival postradiation in medulloblastomas requires YAP, YB1, and IGF2

Sonic hedgehog (Shh)-mediated medulloblastoma growth requires IGF2 (Insulin-like Growth Factor 2) and we recently showed that Yes Associated Protein (YAP1) induces IGF2 expression by Y-box protein 1(YB1) in Shh-stimulated cerebellar granule neural precursors (CGNPs), proposed cells-of-origin for the Shh molecular subclass of medulloblastoma, and mouse Shh-medulloblastoma cells. We found elevated levels of YAP1, YB1 and IGF2 in tumor cells occupying the peri-vascular niche, a microenvironmental niche proposed to house so-called tumor re-populating cells that survive radiation and contribute to medulloblastoma recurrence, which is fatal. We have developed an ex vivo approach using organotypic brain tumor slice cultures to better understand how YAP1, YB1, and IGF2 regulate peri-vascular niche cell survival post-radiation. We observed that the perivascular niche cell population expressing stem cell markers increases markedly following exposure to radiation. Additionally, on targeting any component of the YAP1-YB1-IGF2 axis we observed increased level of cell death within the niche and compromised cancer stem cell niche expansion post-radiation. These findings strongly indicate that therapeutic approaches intended to impair the function of this pathway could be used to reduce the use of cranio-spinal radiation of medulloblastoma patients, which causes life-long side effects that drastically impair quality of life. Citation Format: Abhinav Dey, Caroline Maier, Anshu Malhotra, Anna Kenney. Cancer stem cell survival postradiation in medulloblastomas requires YAP, YB1, and IGF2. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2451.