A simplified approach using Taqman low-density array for medulloblastoma subgrouping

Cruzeiro, Gustavo Alencastro Veiga; Salomão, Karina Bezerra; Biagi, Carlos Alberto Oliveira de; Baumgärtner, Martin; Sturm, Dominik; Lira, Régia Caroline Peixoto; Magalhães, Taciani de Almeida; Baroni, Mirella; Silveira, Vanessa da Silva; Saggioro, Fabiano Pinto; Oliveira, Ricardo Santos de; Klinger, Paulo Henrique dos Santos; Seidinger, Ana Luíza; Yunes, José Andrés; Queiróz, Rosane Gomes de Paula; Oba-Shinjo, Sueli Mieko; Scrideli, Carlos Alberto; Marie, Suely Kazue Nagahashi; Tone, Luíz Gonzaga; Valera, Elvis Terci

doi:10.1186/s40478-019-0681-y

Cited by 23 publications

(18 citation statements)

References 32 publications

(57 reference statements)

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“…The molecular classification was performed by TaqMan Low-Density Array (TLDA PCR-Array). A detailed description of the TDLA method for MB classification can be found elsewhere 23 .…”

Section: Methodsmentioning

confidence: 99%

Arsenic Trioxide exerts cytotoxic and radiosensitizing effects in pediatric Medulloblastoma cell lines of SHH Subgroup

Klinger

Delsin

Cruzeiro

et al. 2020

Sci Rep

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We evaluated the potential effects of ATO in different pediatric SHH-MB cell lines (ONS-76: TP53wild type; DAOY and UW402: TP53-mutated). MB cell lines molecular subgroup was confirmed and TP53 mutations were validated. Cell viability, clonogenicity and apoptosis were evaluated after ATO treatment at different concentrations (1-16 µM) alone or combined with irradiation doses (0.5, 1, 2 and 4 Gy). Rad51 and Ku86 proteins were evaluated by WB. ATO treatment reduced cell viability for all SHH-MB cell lines. Significant decrease of clonogenic capacity and higher apoptosis rates were also observed after ATO exposure, being cell death more pronounced (>70%) for the SHH-MB TP53mutated. Combined treatment of ATO with irradiation also reduced colonies formation in UW402 tumor cells, which was independent of DNA damage repair proteins Rad51 and Ku86. In silico analyses suggested that a set of genes from cell cycle and p53 pathways are differentially expressed in SHH tumor subtypes, suggesting that cell lines may respond to therapies according to the gene expression profiles. Herein, we showed ATO cytotoxicity in pediatric SHH cell lines, with marked radiosensitizing effect for the MB-SHH TP53-mutated cells. These results highlight the potential of ATO, alone or in combination with radiotherapy, supporting further clinical investigations. Medulloblastoma (MB) is the most common malignant brain tumor in children, corresponding to approximately 20% of all brain tumors in patients less than 15 years of age. MB is responsible for significant morbidity and mortality rates 1. Recently, twelve distinct subgroups allocated to four main molecular classes (SHH, WNT, Group 3 and Group 4) have been recognized for MB: WNT-alpha and beta; SHH-alpha, beta, gamma and delta; Group 3-alpha, beta and gamma; and Group 4-alpha, beta, and gamma 2. Considering the clinical significance of the MB molecular classification, new therapies that contemplate the genetic aspects of the disease are required 3. The majority of SHH-MB cases presents somatic mutation in one or more genes of the Sonic Hedgehog (SHH) pathway (i.e., PTCH1, SUFU, or SMO), contributing to its constitutive activation. SHH signaling is essential for embryonic development, as well as it can lead to a tumor arising when aberrantly activated 4. The SHH-MB subgroup prognosis in children is mostly favorable, but the clinical outcome varies within this subgroup. SHH-MB alpha cases are rarely observed in infants and carry the poorest prognosis, which is associated with MYCN and GLI (glioma-associated oncogene homolog) amplification, and higher TP53 mutational burden. In contrast, SHH-MB beta and gamma subtypes affect mainly infants, being one-third of the SHH-MB beta cases metastatic at presentation with frequent focal PTEN deletions. The SHH-MB gamma is strongly related to MB with extensive nodularity (MBEN) histology, in general, presents wild type TP53, and excellent prognosis 2. Lastly,

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“…The molecular classification was performed by TaqMan Low-Density Array (TLDA PCR-Array). A detailed description of the TDLA method for MB classification can be found elsewhere 23 .…”

Section: Methodsmentioning

confidence: 99%

Arsenic Trioxide exerts cytotoxic and radiosensitizing effects in pediatric Medulloblastoma cell lines of SHH Subgroup

Klinger

Delsin

Cruzeiro

et al. 2020

Sci Rep

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“…The human MB cell line DAOY and UW228 (SHH-like subgroup; TP53 mutated—c.725G > T and SHH-like subgroup, c.464C > A, respectively) [ 9 ] was cultured using RPMI-1640 medium supplemented with 10% fetal bovine serum (Gibco BRL, Carlsbad, CA, USA), penicillin (100 U/mL) and streptomycin (100 lg/mL) in a humidified atmosphere with 5% CO 2 at 37 °C. Cell line authentication by short tandem repeats (STR) DNA profiling analysis was performed to assure authenticity and avoid cellular cross-contamination.…”

Section: Methodsmentioning

confidence: 99%

“…Cell line authentication by short tandem repeats (STR) DNA profiling analysis was performed to assure authenticity and avoid cellular cross-contamination. Since DAOY and UW228 are commercially available cell lines and not primary cultures, SHH-like subgroup cell lines are best assigned as SHH subgroups according to comparative transcriptional profiles when compared to medulloblastoma patients [ 9 , 10 , 11 ].…”

Section: Methodsmentioning

confidence: 99%

YAP1 Is a Potential Predictive Molecular Biomarker for Response to SMO Inhibitor in Medulloblastoma Cells

Cruzeiro

Magalhães

Sousa

et al. 2021

Cancers

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Advances in genomics have led to the identification of twelve relevant molecular subtypes within medulloblastoma (MB). The alpha subtype of Sonic hedgehog-driven MB is resistant to therapy (including smoothened inhibitors) due to activation of genes from the non-canonical SHH pathway, such as MYCN, YAP1, or TP53. Using retrospective cohort microarray data, we found that YAP1 is overexpressed in SHH alpha MB and patients profiled as resistant to SMO inhibitors compared to good responders. Here, we performed YAP1 depletion via CRISPR/Cas9 in two in vitro models of SHH-like MB cells and found that this protein is involved in responsiveness to the SMO inhibitor regarding proliferation, apoptosis, and colony formation. Further, considering the synergic combination of YAP1 depletion with SMO inhibition, we assessed single-cell RNA-seq data from five patients and found that SMO and YAP1 are enriched within cells of SHH MB. Importantly, our data suggest that YAP1 is not only a reliable biomarker for cellular response to SMOi but may indicate prospective testing of combination therapy using YAP1 and SMO inhibitors in preclinical models of SHH MB.

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“…Supplemental Table 1 shows the pediatric patients' data. Molecular classi cation was done as previously described [11].…”

Section: Patientsmentioning

confidence: 99%

Integrated microRNA Analysis Identifies miR-512-3p as a Potential Biomarker of Poor Outcome in Pediatric Medulloblastoma

Correia

Chagas

Baroni

et al. 2021

Preprint

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Background: Medulloblastoma, a genetically heterogeneous tumor, is the most frequent malignant brain tumor in children. Although several studies have been carried out, the molecular mechanism underlying medulloblastoma tumorigenesis is not completely known. microRNA (miRNA) expression profiles have been associated with development, progression, and prognosis of human cancers, including medulloblastoma. However, the role of miRNAs in pediatric medulloblastoma has been poorly explored.Methods: Global miRNA expression in 24 microdissected medulloblastoma specimens (19 pediatric and 5 adult specimens) was evaluated by microarray assay. miR-512-3p, the most differentially expressed miRNA in these two groups, was analyzed by qRT-PCR in a cohort of 51 consecutive pediatric medulloblastoma samples and 7 pediatric non-neoplastic cerebellum control samples, and its clinical significance was assessed. Further in silico miRNA prediction of target genes was performed with bioinformatics tools.Results: Compared to the controls, miR-512-3p was significantly downregulated in the pediatric medulloblastoma samples. Higher miR-512-3p was associated with incomplete degree of resection, high risk group classification, and poor overall survival. In silico analysis in an independent cohort of medulloblastoma identified that some of the miR-512-3p target genes (SMAD9, SSX2IP, MAPK10, PTCH1, CCDC6, and BMPR2) were statistically correlated with overall survival, metastasis, and death.Conclusions: For the first time, our results have shown that miR-512-3p is significantly associated with poor clinical outcome in pediatric medulloblastoma, suggesting that miR-512-3p is a potential biomarker of prognosis.

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A simplified approach using Taqman low-density array for medulloblastoma subgrouping

Cited by 23 publications

References 32 publications

Arsenic Trioxide exerts cytotoxic and radiosensitizing effects in pediatric Medulloblastoma cell lines of SHH Subgroup

Arsenic Trioxide exerts cytotoxic and radiosensitizing effects in pediatric Medulloblastoma cell lines of SHH Subgroup

YAP1 Is a Potential Predictive Molecular Biomarker for Response to SMO Inhibitor in Medulloblastoma Cells

Integrated microRNA Analysis Identifies miR-512-3p as a Potential Biomarker of Poor Outcome in Pediatric Medulloblastoma

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