Zsombor Melegh

LGR5 regulates pro-survival MEK/ERK and proliferative Wnt/β-catenin signalling in neuroblastoma

LGR5 is a marker of normal and cancer stem cells in various tissues where it functions as a receptor for R-spondins and increases canonical Wnt signalling amplitude. Here we report that LGR5 is also highly expressed in a subset of high grade neuroblastomas. Neuroblastoma is a clinically heterogenous paediatric cancer comprising a high proportion of poor prognosis cases (~40%) which are frequently lethal. Unlike many cancers, Wnt pathway mutations are not apparent in neuroblastoma, although previous microarray analyses have implicated deregulated Wnt signalling in high-risk neuroblastoma. We demonstrate that LGR5 facilitates high Wnt signalling in neuroblastoma cell lines treated with Wnt3a and R-spondins, with SK-N-BE(2)-C, SK-N-NAS and SH-SY5Y cell-lines all displaying strong Wnt induction. These lines represent MYCN-amplified, NRAS and ALK mutant neuroblastoma subtypes respectively. Wnt3a/R-Spondin treatment also promoted nuclear translocation of β-catenin, increased proliferation and activation of Wnt target genes. Strikingly, short-interfering RNA mediated knockdown of LGR5 induces dramatic Wnt-independent apoptosis in all three cell-lines, accompanied by greatly diminished phosphorylation of mitogen/extracellular signal-regulated kinases (MEK1/2) and extracellular signal-regulated kinases (ERK1/2), and an increase of BimEL, an apoptosis facilitator downstream of ERK. Akt signalling is also decreased by a Rictor dependent, PDK1-independent mechanism. LGR5 expression is cell cycle regulated and LGR5 depletion triggers G1 cell-cycle arrest, increased p27 and decreased phosphorylated retinoblastoma protein. Our study therefore characterises new cancer-associated pathways regulated by LGR5, and suggest that targeting of LGR5 may be of therapeutic benefit for neuroblastomas with diverse etiologies, as well as other cancers expressing high LGR5.

Protein arginine methyltransferase 5 is a key regulator of the MYCN oncoprotein in neuroblastoma cells

Approximately half of poor prognosis neuroblastomas (NBs) are characterized by pathognomonic MYCN gene amplification and MYCN over‐expression. Here we present data showing that short‐interfering RNA mediated depletion of the protein arginine methyltransferase 5 (PRMT5) in cell‐lines representative of NBs with MYCN gene amplification leads to greatly impaired growth and apoptosis. Growth suppression is not apparent in the MYCN‐negative SH‐SY5Y NB cell‐line, or in two immortalized human fibroblast cell‐lines. Immunoblotting of NB cell‐lines shows that high PRMT5 expression is strongly associated with MYCN‐amplification (P < 0.004, Mann–Whitney U‐test) and immunohistochemical analysis of primary NBs reveals that whilst PRMT5 protein is ubiquitously expressed in the cytoplasm of most cells, MYCN‐amplified tumours exhibit pronounced nuclear PRMT5 staining. PRMT5 knockdown in MYCN‐overexpressing cells, including the SHEP‐21N cell‐line with inducible MYCN expression leads to a dramatic decrease in MYCN protein and MYCN‐associated cell‐death in SHEP‐21N cells. Quantitative gene expression analysis and cycloheximide chase experiments suggest that PRMT5 regulates MYCN at a post‐transcriptional level. Reciprocal co‐immunoprecipitation experiments demonstrated that endogenous PRMT5 and MYCN interact in both SK‐N‐BE(2)C and NGP cell lines. By using liquid chromatography – tandem mass spectrometry (LC‐MS/MS) analysis of immunoprecipitated MYCN protein, we identified several potential sites of arginine dimethylation on the MYCN protein. Together our studies implicate PRMT5 in a novel mode of MYCN post‐translational regulation and suggest PRMT5 plays a major role in NB tumorigenesis. Small‐molecule inhibitors of PRMT5 may therefore represent a novel therapeutic strategy for neuroblastoma and other cancers driven by the MYCN oncogene.

Gastrointestinal stromal tumours can express CD10 and epithelial membrane antigen but not oestrogen receptor or HMB45.

Wong N A C S & Melegh Z 
(2011) Histopathology59, 781–785

The small molecule inhibitor YK-4-279 disrupts mitotic progression of neuroblastoma cells, overcomes drug resistance and synergizes with inhibitors of mitosis

Neuroblastoma is a biologically and clinically heterogeneous pediatric malignancy that includes a high-risk subset for which new therapeutic agents are urgently required. As well as MYCN amplification, activating point mutations of ALK and NRAS are associated with high-risk and relapsing neuroblastoma. As both ALK and RAS signal through the MEK/ERK pathway, we sought to evaluate two previously reported inhibitors of ETS-related transcription factors, which are transcriptional mediators of the Ras-MEK/ERK pathway in other cancers. Here we show that YK-4-279 suppressed growth and triggered apoptosis in nine neuroblastoma cell lines, while BRD32048, another ETV1 inhibitor, was ineffective. These results suggest that YK-4-279 acts independently of ETS-related transcription factors. Further analysis reveals that YK-4-279 induces mitotic arrest in prometaphase, resulting in subsequent cell death. Mechanistically, we show that YK-4-279 inhibits the formation of kinetochore microtubules, with treated cells showing a broad range of abnormalities including multipolar, fragmented and unseparated spindles, together leading to disrupted progression through mitosis. Notably, YK-4-279 does not affect microtubule acetylation, unlike the conventional mitotic poisons paclitaxel and vincristine. Consistent with this, we demonstrate that YK-4-279 overcomes vincristine-induced resistance in two neuroblastoma cell-line models. Furthermore, combinations of YK-4-279 with vincristine, paclitaxel or the Aurora kinase A inhibitor MLN8237/Alisertib show strong synergy, particularly at low doses. Thus, YK-4-279 could potentially be used as a single-agent or in combination therapies for the treatment of high-risk and relapsing neuroblastoma, as well as other cancers.

The V600E BRAF mutation in a borderline serous tumour of the testis is genotypic evidence of similarity with its phenotypic ovarian counterpart

mas. They, as well as non-adenomatous background colonic mucosa, demonstrated numerous rounded beads with an untextured magenta appearance similar to that described previously for colestipol. Most beads were in the lumenal surface, but some appeared embedded within the colonic tissue, without evidence of foreign body reaction or other mucosal damage (Figure 1B, C). The striking endoscopic appearance of colestipol granules does not appear to have been reported previously; of the three bile acid sequestrants studied by Arnold et al., colestipol was by far the least commonly encountered. Given that our patient was taking colestipol granules for at least 5 years prior to their gross appearance on colonoscopy, such a finding may simply be a function of prolonged usage. Their relationship, if any, to the development of the numerous tubular adenomas is unknown. Similarly, the rounded shape of the colestipol granules is slightly different from the previously reported appearance of crystalline fragments, which were presumably seen in patients taking the tablet form of colestipol, rather than the granule form. We did not see direct evidence of mucosal injury with the colestipol, unlike the damage observed in some other ingested medications encountered on gastrointestinal biopsy, such as Kayexalate and sevelamer.

DNA content heterogeneity in neuroblastoma analyzed by means of image cytometry and its potential significance

The aim of this study was to determine the frequency and significance of the tumor DNA content heterogeneity in 33 previously untreated human neuroblastomas. We used image cytometry to selectively analyze neuroblasts by excluding karyorrhectic or stromal cells from cytometric measurements. DNA content heterogeneity with more than one clonal subpopulation on DNA histogram was found in 8 of 33 cases. Of these 8 cases, 4 showed MYCN amplification. Double labeling fluorescent in situ hybridization with probes for the centromeric region of chromosome 2 and MYCN gene was used to confirm the DNA content heterogeneity. DNA content heterogeneity was associated with poorer prognosis in this study (P<0.05). There was a significant correlation between euploidy (di- and tetraploidy) and worse prognosis, but only when heterogeneous neuroblastomas with euploid cell population were assigned to euploid tumors (P=0.006). Our results may explain the conflicting data in the literature regarding ploidy and suggest that DNA content heterogeneity and the presence of a euploid population may predict worse prognosis in neuroblastoma patients.

Wnt Signalling Drives Context-Dependent Differentiation or Proliferation in Neuroblastoma

Neuroblastoma is one of the commonest and deadliest solid tumours of childhood, and is thought to result from disrupted differentiation of the developing sympathoadrenergic lineage of the neural crest. Neuroblastoma exhibits intra- and intertumoural heterogeneity, with high risk tumours characterised by poor differentiation, which can be attributable to MYCN-mediated repression of genes involved in neuronal differentiation. MYCN is known to co-operate with oncogenic signalling pathways such as Alk, Akt and MEK/ERK signalling, and, together with c-MYC has been shown to be activated by Wnt signalling in various tissues. However, our previous work demonstrated that Wnt3a/Rspo2 treatment of some neuroblastoma cell lines can, paradoxically, decrease c-MYC and MYCN proteins. This prompted us to define the neuroblastoma-specific Wnt3a/Rspo2-driven transcriptome using RNA sequencing, and characterise the accompanying changes in cell biology. Here we report the identification of ninety Wnt target genes, and show that Wnt signalling is upstream of numerous transcription factors and signalling pathways in neuroblastoma. Using live-cell imaging, we show that Wnt signalling can drive differentiation of SK-N-BE(2)-C and SH-SY5Y cell-lines, but, conversely, proliferation of SK-N-AS cells. We show that cell-lines that differentiate show induction of pro-differentiation BMP4 and EPAS1 proteins, which is not apparent in the SK-N-AS cells. In contrast, SK-N-AS cells show increased CCND1, phosphorylated RB and E2F1 in response to Wnt3a/Rspo2, consistent with their proliferative response, and these proteins are not increased in differentiating lines. By meta-analysis of the expression of our 90 genes in primary tumour gene expression databases, we demonstrate discrete expression patterns of our Wnt genes in patient cohorts with different prognosis. Furthermore our analysis reveals interconnectivity within subsets of our Wnt genes, with one subset comprised of novel putative drivers of neuronal differentiation repressed by MYCN. Assessment of β-catenin immunohistochemistry shows high levels of β-catenin in tumours with better differentiation, further supporting a role for canonical Wnt signalling in neuroblastoma differentiation.

Computer Aided Classification of Neuroblastoma Histological Images Using Scale Invariant Feature Transform with Feature Encoding

Neuroblastoma is the most common extracranial solid malignancy in early childhood. Optimal management of neuroblastoma depends on many factors, including histopathological classification. Although histopathology study is considered the gold standard for classification of neuroblastoma histological images, computers can help to extract many more features some of which may not be recognizable by human eyes. This paper, proposes a combination of Scale Invariant Feature Transform with feature encoding algorithm to extract highly discriminative features. Then, distinctive image features are classified by Support Vector Machine classifier into five clinically relevant classes. The advantage of our model is extracting features which are more robust to scale variation compared to the Patched Completed Local Binary Pattern and Completed Local Binary Pattern methods. We gathered a database of 1043 histologic images of neuroblastic tumours classified into five subtypes. Our approach identified features that outperformed the state-of-the-art on both our neuroblastoma dataset and a benchmark breast cancer dataset. Our method shows promise for classification of neuroblastoma histological images.

Correction: LGR5 regulates pro-survival MEK/ERK and proliferative Wnt/β-catenin signalling in neuroblastoma

Copyright: Vieira et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC-BY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Present: The originally supplied Figure 5 contains duplicate total-ERK panels. Correct: The proper Figure 5 appears below. The authors sincerely apologize for this error. Original article: Oncotarget. 2015; 6:40053-67. doi: 10.18632/oncotarget.5548 Correction

The mutational frequency of BRAF and KRAS in low grade serous testicular neoplasms - a case series.

Low‐grade serous neoplasms of the testis are rare neoplasms that show striking morphological similarities with the better‐understood ovarian neoplasms. This study is to see if there are similar molecular abnormalities in these two tumours. The cell of origin, relationship with serous ovarian tumour and the pathogenesis of these neoplasms are not fully established.