The role of Id2 protein in neuroblatoma in children

Id (DNA binding and/or differentiation) proteins occur physiologically during ontogenesis and negatively regulate the activity of other helix-loop-helix (HLH) proteins. Id2 protein causes block of cells differentiation in the S phase of the cell cycle and regulates the activity of Rb protein. The role of Id2 protein in physiological cell cycle progression and in neuroblastoma (NBL) pathogenesis was proposed by Lasorella. The aim of the study was evaluation of Id2 expression and its prognostic significance in NBL cells coming from primary tumors and evaluation of its prognostic significance, and correlation of Id2 expression with known prognostic factors. Sixty patients with primary NBL treated from 1991 to 2005 were included in the analysis. We found 50 patients with high and 10 patients with low intensity of Id2 expression. The median percentage of NBL cells with Id2 expression was 88 %. We found no correlation between the number of NBL cells or the intensity of Id2 expression and OS and DFS. In patients with stage 4 NBL, almost all patients had high expression of Id2 and it was significantly more common than in other disease stages (p = 0,03). We found no correlation between Id2 expression and other known prognostic factor in NBL patients. We assume that Id2 is not prognostic factor. However, due to its abundant expression in most of NBL cells and its role in cell cycle, it may be potential therapeutic target. Exact knowledge of expression time may be helpful in explaining mechanisms of oncogenesis

ERBB2 as a driver of an invasive phenotype of cells grown in 3D culture and an important regulator of oncogenic miRNAs' expression in breast cancer

Breast cancer is often associated with deregulated activity of two receptor tyrosine kinases (RTKs) from the ERBB family: EGFR and ERBB2 (HER2/Neu). Whereas the EGF receptor is often mutated in breast cancer, ERBB2 is up-regulated in up to 20% of invasive breast tumors and plays a triggering role in cell proliferation, invasion and metastasis. Patients overexpressing ERBB2 are treated with targeted therapies and in particular with the monoclonal antibody trastuzumab, which binds an extracellular domain of the receptor and abrogates receptor-kinase activation. However, ERBB2-positive breast cancer patients overexpress ERBB2 to different extent. The expression range is very wide, raising the question whether the tumors expressing moderate, high and very high ERBB2 levels have different outcome and patients carrying them can be treated as one entity or not. To better understand the impact of different ERBB2 levels I developed stable cell line pools overexpressing ERBB2 at different levels. I observed that in 3D culture as well as in matrigel-based invasion assays, cells expressing very high ERBB2 levels showed more invasive properties than those expressing moderate ERBB2 levels, even in the absence of EGF. This was accompanied by disruption of cell polarity in 3D-grown spheroids and anchorage-independent growth which occured only when ERBB2 expression was very high. These phenotypes could be at least partially explained by ERBB2-dose dependent epithelial-mesenchymal transition (EMT), an increase in HB-EGF transcription and constitutive ERK and AKT signaling pathway activation at the very high ERBB2 levels. As recently several miRNAs have been reported to regulate EMT-related processes and because their expression is frequently deregulated in cancer, I also hypothesized that their expression would change with the ERBB2 expression level in 3D culture. To verify my hypothesis, I performed small RNA sequencing from stable cell line pools grown in matrigel. This identified several miRNAs which are ERBB2-level dependent. I focused then on the functional characterization of miR-301b and miR-130b whose expressions were ERBB2-dependent in 3D, but not in 2D culture. Overexpression of these miRNAs phenocopied ERBB2 effects on cell invasion and proliferation as well as induction of EMT, as did overexpression of the related miR-301a

New method for quantitative analysis of GD2 ganglioside in plasma of neuroblastoma patients

Neuroblastoma, the most common extracranial solid tumour of childhood, is a malignancy of unknown origin and non-specific symptoms. One of the markers of the disease is GD2 ganglioside (disialoganglioside), which is abundantly expressed on the surface of neuroblastoma cells. Gangliosides are known to be shed by tumour cells and this phenomenon can be significant in cancer progression as they inhibit a number of immune responses both in vitro and in vivo. In search for novel markers useful in monitoring and prognosis of neuroblastoma, we developed and validated a new quantitative method of GD2 ganglioside analysis in human blood plasma. We evaluated the level of gangliosides in blood serum of 34 neuroblastoma patients using high-performance liquid chromatography. The technique was used to detect fluorescently labelled oligosaccharides derived from serum glycosphingolipids by enzymatic digestion with ceramide glycanase. The developed method allowed determination of GD2 concentrations at the picomole level and required only 40 µl of plasma, which should be particularly useful when the quantity of clinical material is limiting. Moreover, this method can be applied to study concentration of other gangliosides, as shown for GD3 ganglioside. Analysis of plasma samples from the 34 neuroblastoma patients did not reveal any correlations between the concentration of GD2 ganglioside and clinical parameters, including the results of therapy; it showed, however, that the concentration of GD2 ganglioside in the plasma of neuroblastoma patients decreased substantially in the course of treatment

EGF activates TTP expression by activation of ELK-1 and EGR-1 transcription factors

<p>Abstract</p> <p>Background</p> <p>Tristetraprolin (TTP) is a key mediator of processes such as inflammation resolution, the inhibition of autoimmunity and in cancer. It carries out this role by the binding and degradation of mRNA transcripts, thereby decreasing their half-life. Transcripts modulated by TTP encode proteins such as cytokines, pro-inflammatory agents and immediate-early response proteins. TTP can also modulate neoplastic phenotypes in many cancers. TTP is induced and functionally regulated by a spectrum of both pro- and anti-inflammatory cytokines, mitogens and drugs in a MAPK-dependent manner. So far the contribution of p38 MAPK to the regulation of TTP expression and function has been best described.</p> <p>Results</p> <p>Our results demonstrate the induction of the gene coding TTP (<it>ZFP36</it>) by EGF through the ERK1/2-dependent pathway and implicates the transcription factor ELK-1 in this process. We show that ELK-1 regulates <it>ZFP36 </it>expression by two mechanisms: by binding the <it>ZFP36 </it>promoter directly through ETS-binding site (+ 883 to +905 bp) and by inducing expression of EGR-1, which in turn increases <it>ZFP36 </it>expression through sequences located between -111 and -103 bp.</p> <p>Conclusions</p> <p>EGF activates TTP expression via ELK-1 and EGR-1 transcription factors.</p

Tumor expression of survivin, p53, cyclin D1, osteopontin and fibronectin in predicting the response to neo-adjuvant chemotherapy in children with advanced malignant peripheral nerve sheath tumor

Purpose Selected cell-cycle regulators and extracellular matrix proteins were found to play roles in malignant peripheral nerve sheath tumor (MPNST) biology. We aimed to analyze whether initial tumor tissue expressions of survivin, p53, cyclin D1, osteopontin (OPN) and fibronectin (FN) correlate with the response to neo-adjuvant CHT (naCHT) in children with advanced inoperable MPNST. Methods The study included 26 children with MPNST (M/F 14/12, median age 130 months) treated in Polish centers of pediatric oncology between 1992 and 2013. Tissue expression of markers was studied immunohistochemically in the manually performed tissue microarrays and assessed semi-quantitatively as low and high, based on the rate of positive cells and staining intensity. Results Good response to naCHT was noted in 47.6%, while poor-in 52.4% of patients. The response to naCHT was influenced negatively by the presence of neurofibromatosis NF1 and high initial tumor tissue expression of OPN, survivin, p53 and cyclin D1. Patients with high tumor expression of either OPN, survivin or p53 and those with simultaneous high expression of ≥ 3 of the markers, responded significantly worse to naCHT, than patients, in whom expression of ≤ 2 markers were detected at diagnosis. Nearly, 85% of patients expressing ≥ 3 markers, responded poor to CHT; while 87.5% of children, expressing ≤ 2 markers, were good responders. Conclusion The initial tumor tissue expression of OPN, survivin, p53 and cyclin D1 may serve as markers to predict response to naCHT in pediatric advanced MPNST. Future studies in more numerous group of patients are needed to confirm these preliminary results

Is FLT3 internal tandem duplication an unfavorable risk factor for high risk children with acute myeloid leukemia? : Polish experience

According to the AML-BFM 2004 Interim, a treatment protocol used in Poland since 2005, presence of FLT3 internal tandem duplication (FLT3/ITD) qualifies a patient with acute myeloid leukemia (AML) to a high-risk group (HRG). The present study was aimed to identify the prevalence of FLT3/ITD in children with AML in Poland and to evaluate its prognostic significance in the HRG patients. Out of 291 children with de novo AML treated in 14 Polish centers between January 2006 and December 2012, samples from 174 patients were available for FLT3/ITD analysis. Among study patients 108 children (61.7%) were qualified to HRG. Genomic DNA samples from bone marrow were tested for identification of FLT3/ITD mutation by PCR amplification of exon 14 and 15 of FLT3 gene. Clinical features and treatment outcome in patients with and without FLT3/ITD were analyzed in the study. The FLT3/ITD was found in 14 (12.9%) of 108 HRG children. There were no significant differences between children with and without FLT3/ITD in age and FAB distribution. The white blood cells count in peripheral blood at diagnosis was significantly higher (p <0.01) in the children with FLT3/ITD. Over 5-year overall survival rate for FLT3/ITD positive children was worse (42.4%) comparing to FLT3/ITD negative children (58.9%), but the statistical difference was not significant. However, over 5-year survivals free from treatment failures were similar. The FLT3/ITD rate (12.9%) observed in the study corresponded to the published data. There was no significant impact of FLT3/ITD mutation on survival rates, although further studies are needed on this subject