Deregulated microRNAs in neurofibromatosis type 1 derived malignant peripheral nerve sheath tumors

Malignant peripheral nerve sheath tumors (MPNST) are aggressive cancers that occur spontaneously (sporadic MPNST) or from benign plexiform neurofibromas in neurofibromatosis type 1 (NF1) patients. MPNSTs metastasize easily, are therapy resistant and are frequently fatal. The molecular changes underlying the malignant transformation in the NF1 setting are incompletely understood. Here we investigate the involvement of microRNAs in this process. MicroRNA expression profiles were determined from a series of archival, paired samples of plexiform neurofibroma and MPNST. Ninety differentially expressed microRNAs were identified between the paired samples. Three downregulated microRNAs (let-7b-5p, miR-143-3p, miR-145-5p) and two upregulated microRNAs (miR135b-5p and miR-889-3p) in MPNST were selected for functional characterization. In general, their differential expression was validated in a relevant cell line panel but only partly in a series of unpaired, fresh frozen tumor samples. As part of the validation process we also analyzed microRNA expression profiles of sporadic MPNSTs observing that microRNA expression discriminates NF1-associated and sporadic MPNSTs. The role of microRNAs in cancer progression was examined in NF1-derived MPNST cell lines by transiently modulating microRNA levels. Our findings indicate that some microRNAs affect migratory and invasive capabilities and Wnt signaling activity but the effects are distinct in different cell lines. We conclude that miRNAs play essential regulatory roles in MPNST facilitating tumor progression

Non-Coding RNAs, a Novel Paradigm for the Management of Gastrointestinal Stromal Tumors

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal malignancies found in the gastrointestinal tract. At a molecular level, most GISTs are characterized by gain-of-function mutations in V-Kit Hardy–Zuckerman 4 Feline Sarcoma Viral Oncogene Homolog (KIT) and Platelet Derived Growth Factor Receptor Alpha (PDGFRA), leading to constitutive activated signaling through these receptor tyrosine kinases, which drive GIST pathogenesis. In addition to surgery, treatment with the tyrosine kinase inhibitor imatinib forms the mainstay of GIST treatment, particularly in the advanced setting. Nevertheless, the majority of GISTs develop imatinib resistance. Biomarkers that indicate metastasis, drug resistance and disease progression early on could be of great clinical value. Likewise, novel treatment strategies that overcome resistance mechanisms are equally needed. Non-coding RNAs, particularly microRNAs, can be employed as diagnostic, prognostic or predictive biomarkers and have therapeutic potential. Here we review which non-coding RNAs are deregulated in GISTs, whether they can be linked to specific clinicopathological features and discuss how they can be used to improve the clinical management of GISTs

Patient and tumor characteristics.

<p>Patient and tumor characteristics.</p

Expression level of TOP2A in human neurofibroma and MPNST samples and cell lines.

<p>(<b>A</b>) qRT-PCR was used to determine mRNA levels of <i>TOP2A</i> in paired plexiform neurofibroma (NF, blue, n = 9) and MPNST (red, n = 9) formalin-fixed paraffin-embedded tumor samples, each pair being derived from the same NF1 patient. Asterisk indicates P<0.05. (<b>B</b>) qRT-PCR was used to determine mRNA levels of <i>TOP2A</i> in fresh frozen MPNST (red, n = 11), plexiform neurofibroma (blue, n = 7) and atypical neurofibroma (grey, n = 4). (<b>C</b>) qRT-PCR was used to determine mRNA levels of <i>TOP2A</i> in a cell line panel: Hs53.T neurofibroma cell line (blue) and STS26T, sNF96.2, ST88-14, T265 and 90-8TL MPNST cell lines (red). (<b>D</b>) Western blot displaying TOP2A protein expression in cell line panel and HEK293T. ẞ-actin levels are shown as a loading control.</p

siRNA mediated knockdown of EZH2 and its effect on cell proliferation.

<p>(<b>A</b>) Western blot showing the effect of EZH2 siRNA (si+) or a scrambled control siRNA (si-) on EZH2 protein levels in T265 and 90-8TL at 48h and 72 h post-transfection. (<b>B</b>) Cell proliferation monitored in time after transfection of T265 and 90-8TL with <i>EZH2</i> siRNA (si+) or a scrambled control siRNA (si-). ẞ-actin levels are shown as a loading control.</p

Sensitivity of neurofibroma and MPNST cell lines to the BET bromodomain inhibitor JQ1.

<p>(<b>A</b>) An <i>in vitro</i> cytotoxicity assay (SRB assay) was used to determine IC₅₀ values (nM) for the BET bromodomain inhibitor JQ1 of neurofibroma and MPNST cell lines after a 72h exposure to the drug. Graphs show cell viability as a function of JQ1 concentration. Depicted is the average viability (n = 4) of a representative experiment. (<b>B</b>) Listing of calculated IC₅₀ values and correlation plot, with BRD4 protein expression levels on the Y-axis and IC₅₀ values for JQ1 on the X-axis. Pearson correlation coefficient is depicted in the graph.</p

Sensitivity of neurofibroma and MPNST cell lines to doxorubucin.

<p>(<b>A</b>) An <i>in vitro</i> cytotoxicity assay (SRB assay) was used to determine IC₅₀ values (ng/ml) for doxorubucin of neurofibroma and MPNST cell lines after a 48h exposure to the drug. Graphs show cell viability as a function of doxorubucin concentration. Depicted is the average viability (n = 4) of a representative experiment. (<b>B</b>) Listing of calculated IC₅₀ values and correlation plot, with TOP2A protein expression levels on the Y-axis and IC₅₀ values for doxorubicin on the X-axis. Pearson correlation coefficient is depicted in the graph.</p

Expression level of <i>BRD4</i> in human neurofibroma and MPNST samples and cell lines.

<p>(<b>A</b>) qRT-PCR was used to determine mRNA levels of <i>BRD4</i> in paired plexiform neurofibroma (NF, blue, n = 9) and MPNST (red, n = 9) formalin-fixed paraffin-embedded tumor samples, each pair being derived from the same NF1 patient. Asterisk indicates P<0.05. (<b>B</b>) qRT-PCR was used to determine mRNA levels of <i>BRD4</i> in fresh frozen MPNST (red, n = 11), plexiform neurofibroma (blue, n = 7) and atypical neurofibroma (grey, n = 4). (<b>C</b>) qRT-PCR was used to determine mRNA levels of <i>BRD4</i> in a cell line panel: Hs53.T neurofibroma cell line (blue) and STS26T, sNF96.2, ST88-14, T265 and 90-8TL MPNST cell lines (red) (<b>D</b>) Western blot displaying BRD4 protein expression in cell line panel and HEK293T. ẞ-actin levels are shown as a loading control.</p

Expression level of <i>EZH2</i> in human neurofibroma and MPNST samples and cell lines.

<p>(<b>A</b>) qRT-PCR was used to determine mRNA levels of <i>EZH2</i> in paired plexiform neurofibroma (NF, blue, n = 9) and MPNST (red, n = 9) formalin-fixed paraffin-embedded tumor samples, each pair being derived from the same NF1 patient. Asterisk indicates P<0.05. (<b>B</b>) qRT-PCR was used to determine mRNA levels of <i>EZH2</i> in fresh frozen MPNST (red, n = 11), plexiform neurofibroma (blue, n = 7) and atypical neurofibroma (grey, n = 4). (<b>C</b>) qRT-PCR was used to determine mRNA levels of <i>EZH2</i> in a cell line panel: Hs53.T neurofibroma cell line (blue) and STS26T, sNF96.2, ST88-14, T265 and 90-8TL MPNST cell lines (red). (<b>D</b>) Western blot displaying EZH2 protein expression in cell line panel and HEK293T. ẞ-actin levels are shown as a loading control.</p