Diabetic nephropathy (DN) is the leading cause of chronic kidney disease and end-stage renal failure worldwide. Several mechanisms are involved in the pathogenesis of this disease, which culminate in ...morphological changes such as podocyte injury. Despite the complex diagnosis and pathogenesis, limited attempts have been made to establish new biomarkers for DN. The higher concentration of Mindin protein in the urine of patients with type 2 diabetes mellitus suggests that it plays a role in DN. Therefore, this study investigated whether in situ protein expression of Mindin can be considered a potential DN biomarker. Fifty renal biopsies from patients diagnosed with DN, 57 with nondiabetic glomerular diseases, including 17 with focal segmental glomerulosclerosis (FSGS), 14 with minimal lesion disease (MLD) and 27 with immunoglobulin A nephropathy (IgAN), and 23 adult kidney samples from autopsies (control group) were evaluated for Mindin expression by immunohistochemistry. Podocyte density was inferred by Wilms' tumor 1 (WT1) immunostaining, while foot process effacement was assessed by transmission electron microscopy. Receiver operative characteristic (ROC) analysis was performed to determine the biomarker sensitivity/specificity. Low podocyte density and increased Mindin expression were observed in all cases of DN, regardless of their class. In the DN group, Mindin expression was significantly higher than that in the FSGS, MCD, IgAN and control groups. Higher Mindin expression was significantly positively correlated with foot process effacement only in class III DN cases. Furthermore, Mindin protein presented high specificity in the biopsies of patients with DN (p < 0.0001). Our data suggest that Mindin may play a role in DN pathogenesis and is a promising biomarker of podocyte lesions.
Although ependymoma (EPN) molecular subgroups have been well established by integrated high-throughput platforms, low- and middle-income countries still need low-cost techniques to promptly classify ...these molecular subtypes. Here, we applied low-cost methods to classify EPNs from a Brazilian cohort with 60 pediatric EPN patients. Fusion transcripts (
C11orf95-RELA
,
YAP1-MAMLD1
, and
YAP1-FAM118B
) were investigated in supratentorial EPN (ST-EPNs) samples through RT-PCR/Sanger sequencing and immunohistochemistry (IHC) for p65/L1CAM. qRT-PCR and IHC were used to evaluate expression profiling of
CXorf67
, LAMA2, NELL2, and H3K27me3 in posterior fossa EPN (PF-EPNs) samples. In silico analysis was performed using public microarray data to validate the molecular assignment PF-EPNs with
LAMA2/NELL2
markers. RELA cases and
YAP1-MAMLD1
fusions were identified in nine and four ST-EPNs, respectively. An additional RELA case was identified by IHC. Of note,
LAMA2
and
NELL2
gene expression and immunoprofiling were less accurate for classifying PF-EPNs, which were confirmed by in silico analysis. Yet, H3K27me3 staining was sufficient to classify PF-EPN subgroups. Our results emphasize the feasibility of a simplified strategy to molecularly classify EPNs in the vast majority of cases (49/60; 81.7%). A coordinated combination of simple methods can be effective to screen pediatric EPN with the available laboratory resources at most low-/mid-income countries, giving support for clinical practice in pediatric EPN.
Key messages
Low- and middle-income countries need effective low-cost approaches to promptly distinguish between EPN molecular subgroups.
RT-PCR plus Sanger sequencing is able to recognize the most common types of RELA and YAP1 fusion transcripts in ST-EPNs.
Genetic and protein expressions of LAMA2 and NELL2 are of limited value to accurately stratify PF-EPNs.
Immunohistochemical staining for H3K27me3 may be used as a robust method to accurately diagnose PF-EPNs subgroups.
A coordinated flow diagram based on these validated low-cost methods is proposed to help clinical-decision making and to reduce costs with NGS assessment outside research protocols.
Pediatric adrenocortical tumors (ACT) are rare aggressive neoplasms with heterogeneous prognosis. Despite extensive efforts, identifying reliable prognostic factors for pediatric patients with ACT ...remains a challenge. MicroRNA (miRNA) signatures have been associated with cancer diagnosis, treatment response, and prognosis of several types of cancer. However, the role of miRNAs has been poorly explored in pediatric ACT. In this study, we performed miRNA microarray profiling on a cohort of 37 pediatric ACT and nine nonneoplastic adrenal (NNA) samples and evaluated the prognostic significance of abnormally expressed miRNAs using Kaplan–Meier plots, log‐rank test, and Cox regression analysis. We identified a total of 98 abnormally expressed miRNAs; their expression profile discriminated ACT from NNAs. Among the 98 deregulated miRNAs, 17 presented significant associations with patients’ survival. In addition, higher expression levels of hsa‐miR‐630, ‐139‐3p, ‐125a‐3p, ‐574‐5p, ‐596, ‐564, ‐1321, and ‐423‐5p and lower expression levels of hsa‐miR‐377‐3p, ‐126‐3p, ‐410, ‐136‐3p, ‐29b‐3p, ‐29a‐3p, ‐337‐5p, ‐143‐3p, and 140‐5p were significantly associated with poor prognosis, tumor relapse, and/or death. Importantly, the expression profile of these 17 miRNAs stratified patients into two groups of ACTs with different clinical outcomes. Although some individual miRNAs exhibit potential prognostic values in ACTs, only the 17 miRNA‐based expression clustering was considered an independent prognostic factor for 5‐year event‐free survival (EFS) compared to other clinicopathological features. In conclusion, our study reports for the first time associations between miRNA profiles and childhood ACT prognosis, providing evidence that miRNAs could be useful biomarkers to discriminate patients with favorable and unfavorable clinical outcomes.
Deregulation of the IGF system observed in human tumors indicates a role in malignant cell transformation and in tumor cell proliferation. Although overexpression of the IGF2 and IGF1R genes was ...described in adrenocortical tumors (ACTs), few studies reported their profiles in pediatric ACTs. In this study, the IGF2 and IGF1R expression was evaluated by RT-qPCR according to the patient's clinical/pathological features in 60 pediatric ACT samples, and IGF1R protein was investigated in 45 samples by immunohistochemistry (IHC). Whole transcriptome and functional assays were conducted after IGF1R inhibition with OSI-906 in NCI-H295A cell line. Significant IGF2 overexpression was found in tumor samples when compared with non-neoplastic samples (P<0.001), significantly higher levels of IGF1R in patients with relapse/metastasis (P=0.031) and moderate/strong IGF1R immunostaining in 62.2% of ACTs, but no other relationship with patient survival and clinical/pathological features was observed. OSI-906 treatment downregulated genes associated with MAPK activity, induced limited reduction of cell viability and increased the apoptosis rate. After 24h, the treatment also decreased the expression of genes related to the steroid biosynthetic process, the protein levels of the steroidogenic acute regulatory protein (STAR), and androgen secretion in cell medium, supporting the role of IGF1R in steroidogenesis of adrenocortical carcinoma cells. Our data showed that the IGF1R overexpression could be indicative of aggressive ACTs in children. However, in vitro treatments with high concentrations of OSI-906 (>1μM) showed limited reduction of cell viability, suggesting that OSI-906 alone could not be a suitable therapy to abolish carcinoma cell growth.
Pediatric adrenocortical tumor (ACT) is a rare and aggressive neoplasm, with incidence in southern and southeastern Brazil 10–15 times higher than worldwide. Although microRNAs (miRNAs) have been ...reported to act as tumor suppressors or oncogenes in several cancers, the role of miR-149-3p in ACT remains unknown. In this study, we evaluated the expression of miR-149-3p in 67 pediatric ACT samples and 19 non-neoplastic adrenal tissues. The overexpression of miR-149-3p was induced in H295A cell line, and cell viability, proliferation, colony formation, and cell cycle were assessed by in miR-149-3p mimic or mimic control. In silico analysis were used to predict miR-149-3p putative target genes.
CDKN1A
expression at the mRNA and protein levels was evaluated by qRT-PCR and western blot, respectively. Higher miR-149-3p expression was associated with unfavorable ACT outcomes. Compared to the mimic control, miR-149-3p overexpression increased cell viability and colony formation, and affected cell cycle progression. Also, we identified
CDKN1A
as a potential miR-149-3p target gene, with decreased expression at both the gene and protein levels in miR-149-3p mimic cells. Collectively, these findings suggest that miR-149-3p promotes H295A cell viability by downregulating
CDKN1A
and provide evidence that miR-149-3p may be useful as a novel therapeutic target for pediatric ACT
We evaluated the potential effects of ATO in different pediatric SHH-MB cell lines (ONS-76: TP53-wild 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 TP53-mutated. 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.
Glioblastoma (GBM) is the most common primary malignant neoplasm of the central nervous system and, despite the standard therapy; the patients’ prognoses remain dismal. The miRNA expression profiles ...have been associated with patient prognosis, suggesting that they may be helpful for tumor diagnosis and classification as well as predictive of tumor response to treatment. We described the microRNA expression profile of 29 primary GBM samples (9 pediatric GBMs) and 11 non-neoplastic white matter samples as controls (WM) by microarray analysis and we performed functional in vitro assays on these 2 most differentially expressed miRNAs. Hierarchical clustering analysis showed 3 distinct miRNA profiles, two of them in the GBM samples and a group consisting only of cerebral white matter. When adult and pediatric GBMs were compared to WM, 37 human miRNAs were found to be differentially expressed, with miR-10b-5p being the most overexpressed and miR-630 the most underexpressed. The overexpression of miR-630 was associated with reduced cell proliferation and invasion in the U87 GBM cell line, whereas the inhibition of miR-10b-5p reduced cell proliferation and colony formation in the U251 GBM cell line, suggesting that these miRNAs may act as tumor-suppressive and oncogenic miRNAs, respectively. The present study highlights the distinct epigenetic profiling of adult and pediatric GBMs and underscores the biological importance of mir-10b-5p and miR-630 for the pathobiology of these lethal tumors.
Purposes
Pilocytic astrocytoma (PA) is a low-grade neoplasm frequently found in childhood. PA is characterized by slow growth and a relatively good prognosis. Genetic mechanisms such as activation of ...MAPK, BRAF gene deregulation and neurofibromatosis type 1 (NF1) syndrome have been associated with PA development. Epigenetic signature and miRNA expression profile are providing new insights about different types of tumor, including PAs.
Methods
In the present study we evaluated global miRNA expression in 16 microdissected pediatric PA specimens, three NF1-associated PAs and 11 cerebral white matter (WM) samples by the microarray method. An additional cohort of 20 PAs was used to validate by qRT-PCR the expression of six miRNAs differentially expressed in the microarray data.
Results
Unsupervised hierarchical clustering analysis distinguished one cluster with nine PAs, including all NF1 cases and a second group consisting of the WM samples and seven PAs. Among 88 differentially expressed miRNAs between PAs and WM samples, the most underexpressed ones regulate classical pathways of tumorigenesis, while the most overexpressed miRNAs are related to pathways such as focal adhesion, P53 signaling pathway and gliomagenesis. The PAs/NF1 presented a subset of underexpressed miRNAs, which was also associated with known deregulated pathways in cancer such as cell cycle and hippo pathway.
Conclusions
In summary, our data demonstrate that PA harbors at least two distinct miRNA signatures, including a subgroup of patients with NF1/PA lesions.
Next-generation sequencing platforms are routinely used for molecular assignment due to their high impact for risk stratification and prognosis in medulloblastomas. Yet, low and middle-income ...countries still lack an accurate cost-effective platform to perform this allocation. TaqMan Low Density array (TLDA) assay was performed using a set of 20 genes in 92 medulloblastoma samples. The same methodology was assessed in silico using microarray data for 763 medulloblastoma samples from the GSE85217 study, which performed MB classification by a robust integrative method (Transcriptional, Methylation and cytogenetic profile). Furthermore, we validated in 11 MBs samples our proposed method by Methylation Array 450 K to assess methylation profile along with 390 MB samples (GSE109381) and copy number variations. TLDA with only 20 genes accurately assigned MB samples into WNT, SHH, Group 3 and Group 4 using Pearson distance with the average-linkage algorithm and showed concordance with molecular assignment provided by Methylation Array 450 k. Similarly, we tested this simplified set of gene signatures in 763 MB samples and we were able to recapitulate molecular assignment with an accuracy of 99.1% (SHH), 94.29% (WNT), 92.36% (Group 3) and 95.40% (Group 4), against 97.31, 97.14, 88.89 and 97.24% (respectively) with the Ward.D2 algorithm. t-SNE analysis revealed a high level of concordance (k = 4) with minor overlapping features between Group 3 and Group 4. Finally, we condensed the number of genes to 6 without significantly losing accuracy in classifying samples into SHH, WNT and non-SHH/non-WNT subgroups. Additionally, we found a relatively high frequency of WNT subgroup in our cohort, which requires further epidemiological studies. TLDA is a rapid, simple and cost-effective assay for classifying MB in low/middle income countries. A simplified method using six genes and restricting the final stratification into SHH, WNT and non-SHH/non-WNT appears to be a very interesting approach for rapid clinical decision-making.
Adrenocortical cancer (ACC) is a rare and aggressive type of endocrine tumor with high risk of recurrence and metastasis. The overall survival of patients diagnosed with ACC is low and treatment for ...metastatic stages remain limited to mitotane, which has low efficiency in advanced stages of the disease and is associated with high toxicity. Therefore, identification of new biological targets to improve ACC treatment is crucial. Blockade of the Wnt/beta-catenin pathway decreased adrenal steroidogenesis and increased apoptosis of NCI–H295 human ACC cells, in vitro and in a xenograft mouse model. Aurora kinases play important roles in cell division during the G1-M phase and their aberrant expression is correlated with a poor prognosis in different types of tumors. Hence, we hypothesized that inhibition of aurora kinases activity combined with the beta-catenin pathway blockade would improve the impairment of ACC cell growth in vitro. We studied the combinatorial effects of AMG 900, an aurora kinase inhibitor and PNU-74654, a beta-catenin pathway blocker, on proliferation, survival and tumor progression in multiple ACC cell lines: NCI–H295, CU-ACC1 and CU-ACC2. Exposure of ACC cells to the combination of AMG 900 with PNU-74654 decreased cell proliferation and viability compared to either treatment alone. In addition, AMG 900 inhibited cell invasion and clonogenesis compared to PNU-74654, and the combination showed no greater effects. In contrast, PNU-74654 was more effective in decreasing cortisol secretion. These data suggest that inhibition of aurora kinases activity combined with blockade of the beta-catenin pathway may provide a combinatorial approach for targeting ACC tumors.
•Aurora kinases inhibition increases beta-catenin pathway activation of NCI–H295 cells.•Aurora kinases and Wnt/beta-catenin pathway inhibition decreases adrenocortical cancer cells growth.•Aurora kinase inhibition is efficient in impair NCI–H295 clonogenesis and invasion capacity.•Targeting Aurora kinase A and beta-catenin may be a strategy to treat adrenocortical cancer.
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