Borate Minerals and RNA Stability

The abiotic origin of genetic polymers faces two major problems: a prebiotically plausible polymn. mechanism and the maintenance of their polymd. state outside a cellular environment. The stabilizing action of borate on ribose having been reported, the authors have explored the possibility that borate minerals stabilize RNA. Borate itself does not stabilize RNA. The anal. of a large panel of minerals tested in various phys.-chem. conditions shows that in general no protection is exerted on the RNA backbone, with the interesting exception of ludwigite (Mg2Fe3+BO5). Stability is a fundamental property of nucleic polymers and borate is an abundant component of the planet, hence the prebiotic interest of this anal. L'articolo è disponibile sul sito dell'editore: http://www.mdpi.co

MET inhibition sensitizes rhabdomyosarcoma cells to NOTCH signaling suppression

Rhabdomyosarcoma (RMS) is a pediatric myogenic soft tissue sarcoma. The Fusion-Positive (FP) subtype expresses the chimeric protein PAX3-FOXO1 (P3F) while the Fusion-Negative (FN) is devoid of any gene translocation. FP-RMS and metastatic FN-RMS are often unresponsive to conventional therapy. Therefore, novel therapeutic approaches are needed to halt tumor progression. NOTCH signaling has oncogenic functions in RMS and its pharmacologic inhibition through gamma-secretase inhibitors blocks tumor growth in vitro and in vivo. Here, we show that NOTCH signaling blockade resulted in the up-regulation and phosphorylation of the MET oncogene in both RH30 (FP-RMS) and RD (FN-RMS) cell lines. Pharmacologic inhibition of either NOTCH or MET signaling slowed proliferation and restrained cell survival compared to control cells partly by increasing Annexin V and CASP3/7 activation. Co-treatment with NOTCH and MET inhibitors significantly amplified these effects and enhanced PARP1 cleavage in both cell lines. Moreover, it severely hampered cell migration, colony formation, and anchorage-independent growth compared to single-agent treatments in both cell lines and significantly prevented the growth of FN-RMS cells grown as spheroids. Collectively, our results unveil the overexpression of the MET oncogene by NOTCH signaling targeting in RMS cells and show that MET pathway blockade sensitizes them to NOTCH inhibition

MYOD-SKP2 axis boosts tumorigenesis in fusion negative rhabdomyosarcoma by preventing differentiation through p57Kip2^{Kip2} targeting

Rhabdomyosarcomas (RMS) are pediatric mesenchymal-derived malignancies encompassing PAX3/7-FOXO1 Fusion Positive (FP)-RMS, and Fusion Negative (FN)-RMS with frequent RAS pathway mutations. RMS express the master myogenic transcription factor MYOD that, whilst essential for survival, cannot support differentiation. Here we discover SKP2, an oncogenic E3-ubiquitin ligase, as a critical pro-tumorigenic driver in FN-RMS. We show that SKP2 is overexpressed in RMS through the binding of MYOD to an intronic enhancer. SKP2 in FN-RMS promotes cell cycle progression and prevents differentiation by directly targeting p27$^{Kip1}$ and p57$^{Kip2}$, respectively. SKP2 depletion unlocks a partly MYOD-dependent myogenic transcriptional program and strongly affects stemness and tumorigenic features and prevents in vivo tumor growth. These effects are mirrored by the investigational NEDDylation inhibitor MLN4924. Results demonstrate a crucial crosstalk between transcriptional and post-translational mechanisms through the MYOD-SKP2 axis that contributes to tumorigenesis in FN-RMS. Finally, NEDDylation inhibition is identified as a potential therapeutic vulnerability in FN-RMS

Soma-to-Germline Transmission of RNA in Mice Xenografted with Human Tumour Cells: Possible Transport by Exosomes

Mendelian laws provide the universal founding paradigm for the mechanism of genetic inheritance through which characters are segregated and assorted. In recent years, however, parallel with the rapid growth of epigenetic studies, cases of inheritance deviating from Mendelian patterns have emerged. Growing studies underscore phenotypic variations and increased risk of pathologies that are transgenerationally inherited in a non-Mendelian fashion in the absence of any classically identifiable mutation or predisposing genetic lesion in the genome of individuals who develop the disease. Non-Mendelian inheritance is most often transmitted through the germline in consequence of primary events occurring in somatic cells, implying soma-to-germline transmission of information. While studies of sperm cells suggest that epigenetic variations can potentially underlie phenotypic alterations across generations, no instance of transmission of DNA- or RNA-mediated information from somatic to germ cells has been reported as yet. To address these issues, we have now generated a mouse model xenografted with human melanoma cells stably expressing EGFP-encoding plasmid. We find that EGFP RNA is released from the xenografted human cells into the bloodstream and eventually in spermatozoa of the mice. Tumor-released EGFP RNA is associated with an extracellular fraction processed for exosome purification and expressing exosomal markers, in all steps of the process, from the xenografted cancer cells to the spermatozoa of the recipient animals, strongly suggesting that exosomes are the carriers of a flow of information from somatic cells to gametes. Together, these results indicate that somatic RNA is transferred to sperm cells, which can therefore act as the final recipients of somatic cell-derived information

Characterization of EGFP-expressing A-375 cells.

<p><b>A</b>: EGFP-specific RT-PCR amplification from RNA extracted from whole A-375 cells, either EGFP-infected (lane 1) or non-infected (lane 3), and from the extracellular exosome-containing fraction of infected (lane 5) and non-infected (lane 4) A-375 cells. In lane 2 no RNA was added to the amplification mix. GAPDH was used as standard control. <b>B</b>: PCR amplification of DNA extracted from whole A-375 cells, EGFP-infected (lane 1) and non-infected (lane 4), and from the extracellular exosomal fraction from infected (lane 6) or non-infected (lane 5) A-375; no DNA- and no primer-reactions were loaded for control in lanes 2 and 3, respectively. <b>C</b>: Western immunoblotting analysis of protein extracts from: non-infected A-375 cells (lane 1) and exosomal fraction (lane 2), and from infected A-375 cells (lane 3) and their exosomal fraction (lane 4). CD81 was used as a marker of exosomes.</p

EGFP RNA is present in spermatozoa of mice inoculated with EGFP-infected A-375-cells.

<p><b>A:</b> Murine protamine 2 gene (<i>Prm2</i>) amplification products used to select DNA-free RNA samples. Exemplifying gel of <i>Prm2-</i>specific PCR amplification products of intron-containing DNA from the mouse sperm genome (lane 1) and RT-PCR products from RNA extracted from spermatozoa of non-inoculated (lane 2) and EGFP-expressing A-375⁺ inoculated (lane 3) mice, both showing the spliced <i>Prm2</i> form. <b>B:</b> Southern blot hybridization of RT-PCR amplified RNA from<b>:</b> spermatozoa of non-inoculated control (lane 2) and A-375⁺ inoculated (lane 3) mice, and from non-infected (lane 4) and EGFP-infected (lane 5) A-375 whole cells. Hybridization was carried out with an EGFP-specific internal probe. Lane 1 is a no-RNA control. The bottom panel shows RT-PCR amplification products from the same samples using GAPDH-specific primers as a loading control. <b>C:</b> Southern blot hybridization of RT-PCR amplified RNA from spermatozoa from a control mouse (lane 2) and from a single EGFP-expressing A375⁺ inoculated mouse (lane 3); lane 1 shows a no RNA reaction. As in B, the bottom panel shows GAPDH amplification from the same samples. <b>D:</b> RT-PCR amplification with (+RT) or without (-RT) reverse transcription step of RNA extracted from sperm-depleted epididymis from two inoculated EGFP mice. No EGFP-specific amplification products were visible by ethidium bromide staining (EtBr) nor by Southern blot hybridization (Hyb) using an EGFP radioactive probe. The bottom panel shows GAPDH amplification from the same samples.</p

EGFP-specific RNA in circulating blood from A-375/EGFP-inoculated mice.

<p><b>A:</b> Ethidium bromide staining of specific RT-PCR products from RNA extracted from EGFP-infected A-375 cells (lane 1) and blood-purified extracellular exosomal fraction from inoculated (lane 4) and non-inoculated (lane 5) mice. No RNA and no primers were added to the amplification mix in lanes 2 and 3, respectively. <b>B:</b> EGFP hybridization pattern. The gel in A was blotted on filter, hybridized with ³²P-end labelled EGFP-specific probe, washed and autoradiographed. <b>C</b>: GAPDH-specific amplification products from the same samples.</p