Medulloblastoma Tissues Research Articles

Quantification of microRNAs in Cells and Tissues Using Stem-Loop RT PCR and qPCR.

MicroRNA s are small RNA molecules that regulate gene expression by binding to the 3' untranslated region of the mRNA of their target genes. MicroRNA expression is altered in medulloblastoma as compared to the normal brain and this alteration is often associated with the pathogenesis of this tumor. The quantification of microRNA expression is carried out using quantitative/real-time polymerase chain reaction (PCR). In this chapter, we describe the protocol for the quantification of microRNA s in medulloblastoma tissues and cultured cells. This is carried out in three steps: (1) Extraction of total RNA, (2) Stem-loop reverse-transcriptase PCR, and (3) quantitative PCR.

Hedgehog signaling is controlled by Rac1 activity.

Rationale: The nuclear translocation of transcriptional factor Gli is indispensable for Hedgehog (Hh) signaling activation, whose deregulation causes cancer progressions; however, the mechanisms governing Gli nuclear translocation are poorly understood. Here, we report that the Gli translocation in response to Hh requires Rac1 activation.Methods: C3H10T1/2 cell line and mouse embryonic fibroblasts were used to explore the molecular mechanisms underlying Rac1 activity in regulation of Hh signaling transduction. Transgenic mouse strains and human medulloblastoma (MB) tissue samples were utilized to examine the role of Rac1 in Hh-directed limb bud development and MB progression.Results: We show that upon the binding of Hh to receptor Patched1 (Ptch1), receptor Smoothened (Smo) dissociates from Ptch1 and binds to Vav2, resulting in the increased phosphorylation levels of Vav2 at Y172, which further activates Rac1. The role of Rac1 is dependent on the regulation of phosphorylation levels of KIF3A at S689 and T694, which in turn affects IFT88 stability and subsequently dampens SuFu-Gli complex formation, leading to the release of Gli from the complex and the consequent translocation of Gli into the nucleus. Moreover, Vav2 phospho-Y172 levels are up-regulated in GFAP-Cre;SmoM2+/- mouse cerebellum and human Shh type MB tissues, whereas deficiency of Rac1 in mouse embryonic limb bud ectoderm (Prx1-Cre;Rac1f/f) impedes Hh activation by disruption of Gli nuclear translocation.Conclusion: Together, our results uncover the Rac1 activation and the subsequent Gli translocation as a hitherto uncharacterized mechanism controlling Hh signaling and may provide targets for therapeutic intervention of this signaling pathway.

Multiple Reaction Monitoring-Based Targeted Assays for the Validation of Protein Biomarkers in Brain Tumors.

The emergence of omics technologies over the last decade has helped in advancement of research and our understanding of complex diseases like brain cancers. However, barring genomics, no other omics technology has been able to find utility in clinical settings. The recent advancements in mass spectrometry instrumentation have resulted in proteomics technologies becoming more sensitive and reliable. Targeted proteomics, a relatively new branch of mass spectrometry-based proteomics has shown immense potential in addressing the shortcomings of the standard molecular biology-based techniques like Western blotting and Immunohistochemistry. In this study we demonstrate the utility of Multiple reaction monitoring (MRM), a targeted proteomics approach, in quantifying peptides from proteins like Apolipoprotein A1 (APOA1), Apolipoprotein E (APOE), Prostaglandin H2 D-Isomerase (PTGDS), Vitronectin (VTN) and Complement C3 (C3) in cerebrospinal fluid (CSF) collected from Glioma and Meningioma patients. Additionally, we also report transitions for peptides from proteins – Vimentin (VIM), Cystatin-C (CST3) and Clusterin (CLU) in surgically resected Meningioma tissues; Annexin A1 (ANXA1), Superoxide dismutase (SOD2) and VIM in surgically resected Glioma tissues; and Microtubule associated protein-2 (MAP-2), Splicing factor 3B subunit 2 (SF3B2) and VIM in surgically resected Medulloblastoma tissues. To our knowledge, this is the first study reporting the use of MRM to validate proteins from three types of brain malignancies and two different bio-specimens. Future studies involving a large cohort of samples aimed at accurately detecting and quantifying peptides of proteins with roles in brain malignancies could potentially result in a panel of proteins showing ability to classify and grade tumors. Successful application of these techniques could ultimately offer alternative strategies with increased accuracy, sensitivity and lower turnaround time making them translatable to the clinics.

Novel strategies of Raman imaging for monitoring intracellular retinoid metabolism in cancer cells

We developed a label-free Raman method for whole-cell biochemical imaging to detect molecular processes that occur in normal and cancer brain cells due to retinol transport in human cancers at the level of isolated organelles. Our approach allows to create biochemical maps of retinoids localization in lipid droplets, mitochondria and nuclei in single cells. The maps were capable of discriminating triglycerides (TAG) from retinoids (RE) in lipid droplets (LD), and mitochondria providing an excellent tool to monitor intracellular retinoid metabolism. We detected spectral changes that arose in proteins and lipids due to retinoid metabolism in human cell lines of normal astrocytes and high-grade cancer cells of glioblastoma as well as in human medulloblastoma and glioblastoma tissue. Raman imaging is an effective tool for monitoring retinoids and retinol binding proteins involved in carcinogenesis by detecting unique spectral signatures of vibrations. We found two functionally distinct lipid droplets: TAG-LD, for energy storage, and RE-LD, for regulating mechanisms of signal transduction. Raman polarization measurements revealed the occurrence of conformational changes affecting discrete regions of proteins associated with retinol binding. Aberrant expression of retinoids and retinol binding proteins in human tumours were localized in lipid droplets, and mitochondria.

Molecular Determinants of Medulloblastoma Metastasis and Leptomeningeal Dissemination.

Medulloblastoma is the most common malignant brain cancer in pediatrics consisting of four molecular subgroups, namely wingless (WNT), sonic hedgehog (SHH), Group 3, and Group 4. One of the biggest challenges in the clinical management of this disease is the leptomeningeal dissemination (LMD) of tumor cells with high morbidity and mortality. Many molecular regulators to date have been identified to participate in medulloblastoma metastasis. In the SHH subgroup, the co-upregulation of CXCR4 and PDGFR, as well as the activation of c-MET, show significant promigratory effects on medulloblastoma cells. Amplification or overexpression of genes on the long arm of chromosome 17, such as LASP1 and WIP1, facilitates tumor invasion in both Group 3 and Group 4 medulloblastomas. PRUNE1, NOTCH1, and MYC interactor JPO2 are more specific genetic drivers of metastatic Group 3 tumors. The RAS/MAPK and PI3K/AKT pathways are two crucial signal transduction pathways that may work as the convergent downstream mechanism of various metastatic drivers. Extracellular signals and cellular components in the tumor microenvironment also play a vital role in promoting the spread and colonization of medulloblastoma cells. For instance, the stromal granule cells and astrocytes support tumor growth and dissemination by secreting PlGF and CCL2, respectively. Importantly, the genetic divergence has been determined between the matched primary and metastatic medulloblastoma samples. However, the difficulty of obtaining metastatic medulloblastoma tissue hinders more profound studies of LMD. Therefore, identifying and analyzing the subclone with the metastatic propensity in the primary tumor is essential for future investigation.

Circ-SKA3 upregulates ID3 expression by decoying miR-326 to accelerate the development of medulloblastoma

Medulloblastoma (MB), the most common malignant childhood brain tumor, is a serious threat to life. Circular RNA (circRNA) is involved in the development of various cancers, including MB. We aimed to explore the role of circRNA spindle and kinetochore associated complex subunit 3 (circ-SKA3) in MB progression. Circ-SKA3 expression was elevated in MB tissues and cells. Depleted expression of circ-SKA3 inhibited MB cell proliferation, migration and invasion and induced apoptosis and cell cycle arrest, and circ-SKA3 knockdown inhibited MB growth in vivo. Mechanism analyses revealed that circ-SKA3 directly targeted miR-326 that could bind to ID3, and circ-SKA3 decoyed miR-326 to increasing ID3 expression. Rescue experiments showed that miR-326 inhibition reversed the effects of circ-SKA3 knockdown, and ID3 overexpression recovered MB cell proliferation, migration and invasion blocked by miR-326 restoration. In conclusion, circ-SKA3 functioned as an oncogene to promote the development of MB by increasing ID3 expression via decoying miR-326, hinting that circ-SKA3 might be a therapeutic target of MB.

A systematic view of pediatric medulloblastoma proteomics-current state of the field and future directions.

Quantitative mass spectrometry (MS)-based approaches have allowed further characterization of medulloblastoma (MB) classification and clinical/biological behavior. By investigating protein expression, as well as the role of post-translational modifications in shaping cellular activity, novel avenues of research will clarify the current subgrouping, providing elements for tumor treatment-new molecular targets and signaling cascades-and introducing serum, urinary, and CSF markers of tumor growth and recurrence. We systematically searched and reviewed original research articles treating MB proteomics on PubMed. Reviews, opinion papers, and abstracts were excluded from the final work. A total of 30 novel articles treating the proteomic characterization of MB were included in our review. Research conducted on tissue samples, cell lines, CSF, and urine, as well as exosome and medullospheres, was considered, to picture a broad view of the different directions MS-based proteomic analysis is moving toward. In this review, we collect, summarize, and interpret the current literature on this topic. Significant progress has been achieved in the last decade in MB characterization, paving the way for further exploration of large biobanks of MB and other tissues that will allow a more systematic understanding of MB functioning and clinical progression.

CircSKA3 Modulates FOXM1 to Facilitate Cell Proliferation, Migration, and Invasion While Confine Apoptosis in Medulloblastoma via miR-383-5p.

BackgroundMedulloblastoma (MB) is the most common malignant brain tumor during childhood. Circular RNA (circSKA3) was identified to function as an oncogene in MB. However, the mechanism of circSKA3 in MB remains unclear.MethodsThe levels of circSKA3, microRNA-383-5p (miR-383-5p), and forkhead box M1 (FOXM1) in MB tissues were measured by quantitative real-time polymerase chain reaction (qRT-PCR). The cell viability and apoptotic rate were assessed via 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay and flow cytometry, respectively. The protein levels of B-cell lymphoma 2 (Bcl-2), C-Caspase3, and FOXM1 were detected via Western blot assay. Cell cycle was detected by flow cytometry. The migration and invasion abilities were monitored by Transwell assay. The dual-luciferase reporter assay was constructed to verify the interactions between miR-383-5p and circSKA3 or FOXM1. The mice model experiment was carried out to validate the effects of circSKA3 in vivo.ResultsThe levels of circSKA3 and FOXM1 were significantly elevated, while the level of miR-383-5p was notably declined in MB tissues. CircSKA3 was validated to sponge miR-383-5p, and FOXM1 was a candidate target of miR-383-5p. CircSKA3 silencing impeded cell proliferation, migration, and invasion while promoted apoptosis by targeting miR-383-5p in vitro and retarded xenograft tumor growth in vivo. miR-383-5p suppressed cell proliferation, migration, and invasion but promoted apoptosis in MB cells by regulating FOXM1. CircSKA3 depletion decreased FOXM1 expression via miR-383-5p in MB cells.ConclusionCircSKA3 augmented MB progression partly through miR-383-5p/FOXM1 axis.

Production of a SCID mouse model of medulloblastoma to explore the therapeutic value of targeting tumor driver genes.

Tumor driver genes are genes where structural or sequence mutations confer a selective advantage for cancer cells. The individualized targeting of tumor driver genes has become a topic of interest for tumor treatment. The prognosis for medulloblastoma (MB), a common type of malignant intracranial tumor found in children, is poor. The tumor driver genes and the corresponding targeted drugs remain to be studied. The present study analyzed tumor driver genes from tumor tissue and peripheral blood from one patient with nodular desmoplastic MB with Sonic Hedgehog activation and screened targeted drugs for the tumor driver genes. Additionally, MB tissue was successfully implanted into the SCID mouse which were then used for subsequent drug screening. The present study explored novel treatments for MB from the perspective of tumor driver genes, and may provide new ideas for choosing individualized targeted therapies for patients with MB.

Extracellular vesicle-associated miR-135b and -135a regulate stemness in Group 4 medulloblastoma cells by targeting angiomotin-like 2

BackgroundExtracellular vesicles (EVs) secreted by tumours, including exosomes, are important factors that regulate cell–cell interactions in oncogenesis. Although EV studies are ongoing, the biological understanding of EV-miRNAs derived from brain tumour spheroid-forming cells (BTSCs) of medulloblastoma is poor.PurposesWe explored the specific cellular miRNAs and EV-miRNAs in medulloblastoma BTSCs to determine their potential biological function.MethodsBulk tumor cells (BTCs) and BTSCs were cultured under different conditions from medulloblastoma tissues (N = 10).ResultsTwenty-four miRNAs were simultaneously increased in both cells and EVs derived from BTSCs in comparison to BTCs. After inhibition of miR-135b or miR135a which were the most significantly increased in BTSCs, cell viability, self-renewal and stem cell marker expression decreased remarkably. Through integrated analysis of mRNAs and miRNAs data, we found that angiomotin-like 2 (AMOTL2), which was significantly decreased, was targeted by both miR-135b and miR-135a. STAT6 and GPX8 were targeted only by miR-135a. Importantly, low expression of AMOTL2 was significantly associated with overall poor survival in paediatric Group 3 and Group 4 medulloblastoma patients.ConclusionOur results indicated that inhibition of miR-135b or miR-135a leads to suppress stemness of BTSC through modulation of AMOTL2.

MiR-30b-5p inhibits proliferation and promotes apoptosis of medulloblastoma cells via targeting MYB proto-oncogene like 2 (MYBL2)

Medulloblastoma (MB) is the most common malignant brain tumors among children. MiR-30b-5p is a potential tumor suppressor in a variety of human cancers. However, its expression and function in MB...

FHOD3 promotes carcinogenesis by regulating RhoA/ROCK1/LIMK1 signaling pathway in medulloblastoma.

Medulloblastoma (MB) is a malignant brain disease in young children. The overall survival of MB patients is disappointing due to absence of effective therapeutics and this could be attributed to the lack of molecular mechanism underlying MB. FHOD3 was an important gene during cardio-genesis and was reported to promote cell migration in cancer. However, its role in MB is not clear to date. RT-qPCR and IHC analysis were used to determine expression of FHOD3. Survival curve was drawn by K-M analysis. FHOD3 was knocked down by RNAi technology. The effects of FHOD3 on medulloblastoma cells were determined by CCK-8 assay, colony formation assay, transwell assay and FACs analysis. FHOD3 expression increased by 1.5 fold in tumor tissues compared to the control and IHC analysis further confirmed strong expression of FHOD3 in medulloblastoma tissues. Then higher FHOD3 expression was associated with shorter survival time in MB patients (13.0months versus 43.8months). In medulloblastoma cells such as Daoy and D283med, FHOD3 also displayed abundant expression. When FHOD3 was knocked down, the ability of cell proliferation and colony formation was reduced over greatly. The capability of cell migration and invasion was also inhibited significantly. However, cell apoptotic rate increased significantly reversely. Mechanistically, the phosphorylation level of RhoA, ROCK1, and LIMK1 was decreased when FHOD3 was knocked down but increased reversely when FHOD3 was over-expressed in Daoy cells. FHOD3 was associated with overall survival time in medulloblastoma patients and was essential to cell proliferation, growth and survival in medulloblastoma and might regulates activation of RhoA/ROCK1/LIMK1 signaling pathway.

Long noncoding RNA HOTAIR promotes medulloblastoma growth, migration and invasion by sponging miR-1/miR-206 and targeting YY1

PurposeLong non-coding RNA (LncRNA) HOX transcript antisense RNA (HOTAIR) and Yin Yang 1 (YY1) are reported to be involved in tumorigenesis. However, the effect and molecular mechanism of HOTAIR on YY1 expression remains poorly understood. The study aimed to investigate the functions and molecular mechanism of LncRNA HOTAIR in medulloblastoma progression. MethodsqPCR was performed to detect HOTAIR and YY1 mRNA in tissues and cells, as well as that of miR-1 and miR-206 expression levels. Western blot assay was used to test YY1 and EMT-related biomarkers’ protein levels. Cell proliferation was tested with CCK-8 assay and colony formation assay. Migration and invasion abilities were tested with Transwell migration and invasion assays. Tumor growth was tested with an in vivo animal study. Cell apoptosis was tested with an Annexin V-FITC/PI kit. Luciferase assay was used to test the luciferase intensity of YY1 and HOTAIR. RNA pull down assay was used to detect the combination between HOTAIR and miR-1/miR-206. ResultsIn this study, we found that HOTAIR and YY1 were up-regulated in medulloblastoma tissues and cell lines, and HOTAIR increased YY1 expression. The molecular mechanism demonstrated that HOTAIR negatively regulated miR-1 and miR-206 expression, which can directly target YY1 in medulloblastoma cells. Moreover, HOTAIR increased YY1 expression through binding to miR-1 and miR-206. The functional experiments showed that HOTAIR knockdown suppressed medulloblastoma cell proliferation, tumor growth, migration and invasion, and promoted cell apoptosis via the modulation of the miR-1/miR-206-YY1 axis, as well as epithelial to mesenchymal transition (EMT). ConclusionThese data indicate that HOTAIR promotes medulloblastoma progression via acting as a competing endogenous RNA (ceRNA) to regulate YY1 expression through binding to miR-1 and miR-206.

PELP1 signaling contributes to medulloblastoma progression by regulating the NF-κB pathway.

Medulloblastoma (MB) is the most common and deadliest brain tumor in children. Proline-, glutamic acid-, and leucine-rich protein 1 (PELP1) is a scaffolding protein and its oncogenic signaling is implicated in the progression of several cancers. However, the role of PELP1 in the progression of MB remains unknown. The objective of this study is to examine the role of PELP1 in the progression of MB. Immunohistochemical analysis of MB tissue microarrays revealed that PELP1 is overexpressed in the MB specimens compared to normal brain. Knockdown of PELP1 reduced cell proliferation, cell survival, and cell invasion of MB cell lines. The RNA-sequencing analysis revealed that PELP1 knockdown significantly downregulated the pathways related to inflammation and extracellular matrix. Gene set enrichment analysis confirmed that the PELP1-regulated genes were negatively correlated with nuclear factor-κB (NF-κB), extracellular matrix, and angiogenesis gene sets. Interestingly, PELP1 knockdown reduced the expression of NF-κB target genes, NF-κB reporter activity, and inhibited the nuclear translocation of p65. Importantly, the knockdown of PELP1 significantly reduced in vivo MB progression in orthotopic models and improved the overall mice survival. Collectively, these results suggest that PELP1 could be a novel target for therapeutic intervention in MB.

SCIDOT-41. NANOTHERAPEUTIC TARGETING OF TUMOR ENDOTHELIUM FOR ENHANCING DRUG DELIVERY PAST THE BLOOD-BRAIN BARRIER

Abstract OBJECTIVE The Sonic Hedgehog (SHH) medulloblastoma subgroup accounts for ~25% of all cases and has an intermediate prognosis. Current conventional therapies result in devastating morbidities including intellectual disability and secondary malignancies. Although molecularly targeted agents that inhibit the SHH pathway have demonstrated clinical efficacy, recent studies have shown on-target secondary toxicities on bone development suggesting new therapeutic approaches are needed. METHODS We investigated the efficacy of the SHH pathway inhibitor, Vismodegib packaged in a fucoidan-based nanoparticle (Fi-Vis) that targets P-selectin, a protein overexpressed on vascular endothelial cells and induced by low-dose ionizing radiation (XRT) in a time- and dose-dependent manner. This P-selectin targeting nanoparticle drug delivery system shows selectivity toward tumor vasculature and not normal brain vasculature in a genetic SHH medulloblastoma mouse model as assessed by ex vivo infrared imaging and two-photon intravital imaging. RESULTS Quantitative RT-PCR analysis of SHH medulloblastoma tissue following single dose XRT and Fi-Vis treatment (10mg/kg) showed a synergistic inhibition of Gli1 expression (>90% target inhibition). Furthermore, we demonstrate that very low dose XRT (0.25Gy) can induce P-selectin expression specifically within MB tumor vasculature and synergize with low dose Fi-Vis (10mg/kg) to significantly enhance mouse survival (p<0.01) when compared to radiation or Fi-Vis alone. Furthermore, assessment of bone toxicity using micro-CT and histological analysis following Fi-Vis administration in postnatal (P10) mice shows no bone toxicity when compared to free Vismodegib. Finally, in vitro studies using mouse brain endothelial cells suggest at least in part a caveolin-1 mediated transcytosis mechanism of crossing the endothelial blood-brain barrier. CONCLUSIONS These data suggest applicability of combined XRT and tumor vasculature-targeted nanotherapeutic dose de-escalation strategies for SHH medulloblastoma with implications for other pediatric and adult brain tumors.

Long Noncoding RNA TP73-AS1 Modulates Medulloblastoma Progression In Vitro And In Vivo By Sponging miR-494-3p And Targeting EIF5A2.

BackgroundPrevious studies have shown that P73 antisense RNA 1T (non-protein coding), also known as TP73-AS1, is a long non-coding RNA (lncRNA) and involved in the development of medulloblastoma. However, the regulatory mechanism of lncRNA TP73-AS1 in medulloblastoma was still unclear, the present study was aimed to investigate the detailed functions and the mechanism of TP73-AS1 in regulation of medulloblastoma.Materials and methodsThe levels of TP73-AS1, miR-494-3p, and Eukaryotic initiation factor 5A2 (EIF5A2) were determined using quantitative real-time PCR (qRT-PCR), in situ hybridization (ISH), or Immunohistochemistry (IHC). The function of TP73-AS1 in proliferation, apoptosis, migration, and invasion of medulloblastoma cells was evaluated using cell counting Kit-8 (CCK-8), flow cytometry, and transwell assay, respectively. The protein levels were determined by Western blot. Bioinformatics analysis and dual-luciferase reporter assay, RNA immunoprecipitation (RIP) and pull-down assay were used to search and confirm the target gene of TP73-AS1 and miR-494-3p. The effect of TP73-AS1 knockdown in vivo was detected by animal experiment.ResultsThe levels of TP73-AS1 and EIF5A2 were up-regulated, while miR-494-3p expression was down-regulated in medulloblastoma tissues and cells, ELF5A2 was a direct target of miR-494-3p, and miR-494-3p bound to TP73-AS1. The knockdown of TP73-AS1 inhibited cell proliferation, invasion, migration, and promoted apoptosis of medulloblastoma cells, while the miR-494-3p inhibitor abolished the effects of TP73-AS1 knockdown on medulloblastoma cells.ConclusionTP73-AS1 positively regulated EIF5A2 expression by sponging miR-494-3p. These findings suggested that TP73-AS1 served as an oncogene and promoted the progression of medulloblastoma.

Protein Kinase A Distribution in Meningioma.

Deregulation of intracellular signal transduction pathways is a hallmark of cancer cells, clearly differentiating them from healthy cells. Differential intracellular distribution of the cAMP-dependent protein kinases (PKA) was previously detected in cell cultures and in vivo in glioblastoma and medulloblastoma. Our goal is to extend this observation to meningioma, to explore possible differences among tumors of different origins and prospective outcomes. The distribution of regulatory and catalytic subunits of PKA has been examined in tissue specimens obtained during surgery from meningioma patients. PKA RI subunit appeared more evenly distributed throughout the cytoplasm, but it was clearly detectable only in some tumors. RII was present in discrete spots, presumably at high local concentration; these aggregates could also be visualized under equilibrium binding conditions with fluorescent 8-substituted cAMP analogues, at variance with normal brain tissue and other brain tumors. The PKA catalytic subunit showed exactly overlapping pattern to RII and in fixed sections could be visualized by fluorescent cAMP analogues. Gene expression analysis showed that the PKA catalytic subunit revealed a significant correlation pattern with genes involved in meningioma. Hence, meningioma patients show a distinctive distribution pattern of PKA regulatory and catalytic subunits, different from glioblastoma, medulloblastoma, and healthy brain tissue. These observations raise the possibility of exploiting the PKA intracellular pathway as a diagnostic tool and possible therapeutic interventions.

High expression of the transcriptional coactivator TAZ is associated with a worse prognosis and affects cell proliferation in patients with medulloblastoma.

The transcriptional coactivator tafazzin (TAZ) serves pivotal roles in organ development, tumor initiation and tumor progression. However, to the best of our knowledge, the expression of TAZ and its clinical significance in human medulloblastoma have not been defined. The present study aimed to clarify the clinical and biological significance of TAZ expression in human medulloblastoma. Immunohistochemical staining for TAZ was performed with 72 medulloblastoma and three normal brain tissue samples. A high expression level of TAZ was detected in 65.28% of medulloblastoma tissues, whereas low expression was identified in the normal brain tissues. TAZ expression was significantly associated with medulloblastoma recurrence. However, the expression of TAZ was not associated with sex, age, tumor location, tumor maximal diameter and tumor histology. Furthermore, both the overall survival and tumor-free survival rate of patients with high levels of expression of TAZ were shorter compared with those of patients with tumors expressing low levels of TAZ. In univariate and multivariate Cox regression analyses, TAZ expression was identified as a significant prognostic factor for patients with medulloblastoma. Functionally, downregulation of TAZ inhibited the proliferation and tumor formation of medulloblastoma cells and the expression of cell-cycle associated proteins in Daoy cells. In conclusion, high expression of TAZ may serve as a prognostic marker for patients with medulloblastoma and TAZ may be a potential target for medulloblastoma therapy.

Abstract 3485: PELP1 is a novel mediator of medulloblastoma progression

Abstract Background: Medulloblastoma (MB) is the most common and deadliest primary brain tumor in children that accounts for 15-20% of all pediatric brain tumors. Despite recent advances in multimodal treatment, the 5-year overall survival of MB patients is approximately 60-70%. Unfortunately, improved outcome have been associated with significant long-term toxicities. Identifying novel targets that drive MB progression is urgently needed. Proline-, glutamic acid-, and leucine-rich protein 1 (PELP1) is a scaffolding protein that functions as a coregulator of several nuclear receptors. Oncogenic PELP1 signaling is implicated in the progression of several cancers including breast, ovarian, prostate, lung, pancreas and colon. However, its role in the progression of medulloblastoma remains unknown. Here, we examined the role of proto-oncogene PELP1 in the progression of MB. Methods: The expression of PELP1 in tumor micro arrays was analyzed using validated PELP1 antibodies for immunohistochemistry. PELP1 expression was determined in vitro by western blotting. PELP1 knockdown cells were generated using PELP1 shRNA lentiviral particles or PELP1 siRNA. The effect of PELP1 knockdown or overexpression was studied using cell proliferation, colony formation and migration using established in vitro assays. Mechanistic studies were conducted using RNA-seq, RT-qPCR, immunohistochemistry, reporter gene assays and signaling analysis. Interaction of PELP1 with NF-κB was examined by immunoprecipitation. Mouse orthotopic xenografts models were used for preclinical evaluation of PELP1 knock down. Results: Immunohistochemical analysis of MB tissue microarrays revealed that PELP1 is overexpressed in MB specimens compared to normal brain specimens. Knockdown of PELP1 using PELP1 specific siRNA or shRNA significantly reduced cell proliferation, cell survival, and cell migration of MB cell lines. RNA-seq analysis revealed that PELP1 knockdown significantly downregulated the pathways related to inflammation, angiogenesis and extracellular matrix. Further, gene set enrichment analysis (GSEA) confirmed that the PELP1-regualted genes were negatively correlated with NF-κB, extracellular matrix and angiogenesis. Mechanistic studies showed that PELP1 knockdown reduced the expression of NF-κB reporter gene activity and its target genes. Additionally, knockdown of PELP1 significantly reduced in vivo MB tumor progression in orthotopic models, and improved overall mice survival. IHC analysis demonstrated that the proliferation marker Ki67 and NF- κB targets were significantly downregulated in PELP1 knockdown tumors compared to controls. Conclusions: Taken together, these results provide the evidence that PELP1 could be a potential therapeutic target for therapeutic intervention in medulloblastoma. Citation Format: Yiliao Luo, Gangadhara Reddy Sareddy, Uday P. Pratap, Mengxing Li, Junhao Liu, Prabhakar Pitta-Venkata, Suryavathi Viswanadhapalli, Xiaonan Li, Manjeet Rao, Rajeshwar Rao Tekmal, Ratna K. Vadlamudi. PELP1 is a novel mediator of medulloblastoma progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3485.

Abstract 3660: A link between miRNAs and mRNA translation elongation: The let7-eEF2K axis in MYC-driven pediatric tumors adaptation to nutrient deprivation

Abstract BACKGROUND/OBJECTIVES: MYC family proteins are implicated in many human cancers, but their therapeutic targeting has proven challenging. MYCN and MYC amplification in childhood neuroblastoma (NB) and medulloblastoma (MB) are associated with aggressive disease and high mortality, underscoring a dire need for novel therapies. Let-7 microRNAs (miRNAs) inhibit tumor progression and regulate metabolism by degrading several mRNAs, including MYC. Let-7 miRNAs are therefore frequently repressed in cancer, including MYC-driven NB and MB. We previously reported that the mRNA translation elongation regulator eukaryotic Elongation Factor-2 Kinase (eEF2K) is a pivotal mediator of cancer cells adaptation to nutrient deprivation (ND). Publicly available transcriptomic database analyses indicate that eEF2K expression significantly correlate with MYCN and MYC expression in multiple tumor cohorts. Our preliminary data also indicate that the eEF2K 3’ untranslated region (UTR) harbors a potential binding site for let-7 miRNAs. In addition, eEF2K mRNA and let-7 miRNA expression negatively correlates in NB and MB, suggesting a potential regulation of the former by the latter. We therefore hypothesized that let-7 down-regulation induces eEF2K expression, thereby supporting MYC-driven NB and MB adaptation to ND and tumor progression. METHODS: Immunohistochemistry for eEF2K substrate (p-eEF2) was performed on NB and MB tissue microarrays to link results with MYC expression and outcome. Effects of eEF2K pharmacological and genetic inhibition on NB and MB cell survival were evaluated in vitro by MTT assay and PI staining. The ability of let-7 to degrade eEF2K mRNA was assessed by let-7 miRNAs transfection into MB cells, followed by RT-PCR and Western Blotting for eEF2K. Binding of let-7 to the eEF2K 3’UTR was validated by luciferase reporter assay. Finally, NB xenograft mouse models were used to confirm in vitro observations. RESULTS: High eEF2K activity is linked to MYC over-expression and reduced survival in NB and MB (p<0.05). Pharmacological and genetic inhibition of eEF2K significantly reduces survival of MYC/MYCN-amplified NB and MB cell lines under ND. Let-7 miRNAs transfection decreases eEF2K mRNA and protein levels (by ~40-50%), and down-regulation of luciferase activity by let-7 miRNAs is impaired upon mutation of the let-7 binding site on the eEF2K 3’UTR. Knockdown of eEF2K determines a twofold growth decrease of MYCN-amplified NB xenografts when mice are kept under caloric restriction diet. CONCLUSIONS: Let-7 miRNAs degrade eEF2K mRNA by binding to its 3'UTR, indicating that let-7 repression in MYC-driven NB and MB is partially responsible for eEF2K increased levels and activity. Moreover, the let-7-eEF2K axis represents a critical mechanism for MYC-driven NB and MB adaptation to ND, constituting a promising therapeutic target. Citation Format: Alberto Delaidelli, Gian Luca Negri, Brian Cho, Simran Sidhu, Stefan Pfister, Michael Taylor, Gabriel Leprivier, Marcel Kool, Poul Soresnsen. A link between miRNAs and mRNA translation elongation: The let7-eEF2K axis in MYC-driven pediatric tumors adaptation to nutrient deprivation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3660.