Abstract Vascular Endothelial Growth Factor A (VEGFA) is the most commonly expressed angiogenic growth factor in solid tumors and is generated as multiple isoforms through alternative mRNA splicing. Here, we show that lncRNA MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) and ID4 (inhibitor of DNA-binding 4) protein, previously referred to as regulators of linear isoforms of VEGFA , induce back-splicing of VEGFA exon 7, producing circular RNA circ_0076611. Circ_0076611 is detectable in triple-negative breast cancer (TNBC) cells and tissues, in exosomes released from TNBC cells and in the serum of breast cancer patients. Circ_0076611 interacts with a variety of proliferation-related transcripts, included MYC and VEGFA mRNAs, and increases cell proliferation and migration of TNBC cells. Mechanistically, circ_0076611 favors the expression of its target mRNAs by facilitating their interaction with components of the translation initiation machinery. These results add further complexity to the multiple VEGFA isoforms expressed in cancer cells and highlight the relevance of post-transcriptional regulation of VEGFA expression in TNBC cells.
During hematopoiesis it is clearly emerging that microRNAs are integrated with target genes in regulatory circuitries involved in the decisions regarding the ability to self-renew and to generate a differentiated progeny in hematopoietic cells including myeloid cells (1). microRNAs are able to modulate genes expression mainly by tuning the rate of proteins’ synthesis inhibiting the initiation or later stages of translation. A change in the association of an mRNA with polysomes is indicative of changes in its translation state. For instance, a block in translational initiation would result in reduced ribo- some density on the affected mRNA and a shift toward the lower-density fractions of the gradient (2). Prediction algorithms usually provide hundreds of target genes for each microRNA and the identification of reliable target genes is feasible only through single-gene approaches. To identify microRNA-mRNA networks relevant for the transition from myeloblasts to monocytes, we here evaluated the localization of microRNAs and mRNAs in ribosomal/polysomal cell fractions obtained by sucrose density gradient centrifugation from the myeloblastic cell line HL60 induced or not to differentiate by 1,25-dihydroxyvitamin D3 treatment to induce monocyte/macrophage differentiation. The co-localization of miRNAs and predicted target mRNAs in low-density riboso- mal fractions is strongly indicative of their functional interaction. Intersection between mRNAs shifted across the fractions after treatment with putative target genes of modulated microRNAs showed a series of molecular net- works relevant for the monocyte cell fate determination, as for example the post-tran- scriptional regulation of the Polo-like kinase 1 (PLK1) by miR-22-3p and let-7e-5p. The disclosing of new molecular players involved in myeloid cell fate determina- tion paves the way for the identification of new potentially interesting molecular tar- gets for the treatment of acute myeloid leukemia cells.
Abstract Background In recent years, the use of ferritins as nano-vehicles for drug delivery is taking center stage. Compared to other similar nanocarriers, Archaeoglobus fulgidus ferritin is particularly interesting due to its unique ability to assemble-disassemble under very mild conditions. Recently this ferritin was engineered to get a chimeric protein targeted to human CD71 receptor, typically overexpressed in cancer cells. Results Archaeoglobus fulgidus chimeric ferritin was used to generate a self-assembling hybrid nanoparticle hosting an aminic dendrimer together with a small nucleic acid. The positively charged dendrimer can indeed establish electrostatic interactions with the chimeric ferritin internal surface, allowing the formation of a protein-dendrimer binary system. The 4 large triangular openings on the ferritin shell represent a gate for negatively charged small RNAs, which access the internal cavity attracted by the dense positive charge of the dendrimer. This ternary protein-dendrimer-RNA system is efficiently uptaken by acute myeloid leukemia cells, typically difficult to transfect. As a proof of concept, we used a microRNA whose cellular delivery and induced phenotypic effects can be easily detected. In this article we have demonstrated that this hybrid nanoparticle successfully delivers a pre-miRNA to leukemia cells. Once delivered, the nucleic acid is released into the cytosol and processed to mature miRNA, thus eliciting phenotypic effects and morphological changes similar to the initial stages of granulocyte differentiation. Conclusion The results here presented pave the way for the design of a new family of protein-based transfecting agents that can specifically target a wide range of diseased cells. Graphic abstract
The clonal expansion of hematopoietic myeloid precursors blocked at different stages of differentiation characterizes the acute myeloid leukemia (AML) phenotype. A subtype of AML, acute promyelocytic leukemia (APL), characterized by the chimeric protein PML-RARα is considered a paradigm of differentiation therapy. In this leukemia subtype the all-trans-retinoic acid (RA)-based treatments are able to induce PML-RARα degradation and leukemic blast terminal differentiation [1-2]. Granulocytic differentiation of APL cells driven by RA triggers a physiological Unfolded Protein Response (UPR), a series of pathways emanating from the ER in case of ER stress, which ensues when higher protein folding activity is required as during differentiation. We show here that, although mild, the ER stress induced by RA is sufficient to render human APL cell lines and primary blasts very sensitive to low doses of Tunicamycin (Tm), an ER stress inducing drug, at doses that are not toxic in the absence of RA. Importantly only human progenitors cells derived from APL patients resulted sensitive to the combined treatment with RA and Tm whereas those obtained from healthy donors were not affected. We also show that the UPR pathway downstream of PERK plays a major protective role against ER stress in differentiating cells and, by using a specific PERK inhibitor, we potentiated the toxic effect of the combination of RA and Tm. In conclusion, our findings identify the ER stress-related pathways as potential targets in the search for novel therapeutic strategies in AML.
The highly conserved Argonaute protein family members play a central role in the regulation of gene expression networks, orchestrating the establishment and the maintenance of cell identity throughout the entire life cycle, as well as in several human disorders, including cancers. Four functional Argonaute proteins (AGO1-4), with high structure similarity, have been described in humans and mice. Interestingly, only AGO2 is robustly expressed during human and mouse early development, in contrast to the other AGOs. Consequently, AGO2 is indispensable for early development in vivo and in vitro. Here, we review the roles of Argonaute proteins during early development by focusing on the interplay between specific domains of the protein and their function. Moreover, we report recent works highlighting the importance of AGO post-translational modifications in cancer.
Abstract Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm caused by the presence of tyrosine kinase BCR-ABL1 fusion protein, which deregulate transcription and mRNA translation. Tyrosine kinase inhibitors (TKIs) are the first-choice treatment. However, resistance to TKIs remains a challenge to cure CML patients. Here, we reveal that the m 6 A methyltransferase complex METTL3/METTL14 is upregulated in CML patients and that is required for proliferation of primary CML cells and CML cell lines sensitive and resistant to the TKI imatinib. We demonstrate that depletion of METTL3 strongly impairs global translation efficiency. In particular, our data show that METTL3 is crucial for the expression of genes involved in ribosome biogenesis and translation. Specifically, we found that METTL3 directly regulates the level of PES1 protein identified as an oncogene in several tumors. We propose a model in which nuclear METTL3/METTL14 methyltransferase complex modified nascent transcripts whose translation is enhanced by cytoplasmic localization of METTL3, independently from its catalytic activity. In conclusion, our results point to METTL3 as a novel relevant oncogene in CML and as a promising therapeutic target for TKI resistant CML.
ABSTRACT Background Phosphodiesterases (PDEs) are the enzymes that hydrolyze cyclic nucleotides (cAMP and cGMP) playing a key role in the homeostasis of these two second messengers. PDE2A is a dual-specific PDE that breaks down both cAMP and cGMP and can be activated by cGMP. It appears peculiar that the Pde2A-deficient ( Pde2A -/- ) mouse model is embryonically lethal, likely due to a strongly reduced size of liver and to a severe anemia. In addition, the heart of Pde2A -/- embryos shows ventricular and atrial septum defects, hypertrabeculation, heart dilatation and non-compaction defects. We recently highlighted a direct relationship between Pde2A impairment, consequent increase of cAMP and the onset of mouse congenital heart defects (CHDs), however the molecular mechanisms underlining the heart defects remain unknown. Methods Transcriptome analysis of Pde2A -/- embryonic heart was performed by RNA sequencing and the most altered genes were also analyzed by quantitative real time PCR. In vivo treatment with drugs acting on cAMP signaling (Metoprolol and H89) and oxidative stress (N-Acetyl-Cysteine, NAC) were carried out on pregnant Pde2A +/- female. Histological, biochemical, and molecular analyses were then performed on embryonic hearts. Results We found a significant modulation of more than 500 genes affecting biological processes involved in the immune system, cardiomyocyte development and contractility, angiogenesis, control of gene transcription and oxidative stress in hearts from Pde2A -/- embryos. Metoprolol and H89 administration were able to prevent heart dilatation and hypertabeculation in Pde2A -/- embryos. Metoprolol was also able to partially impede heart septum defect and oxidative stress at tissue and molecular levels. Partial rescue of cardiac defects was observed by using the antioxidant NAC, indicating oxidative stress like one of the molecular mechanisms underpinning the CHDs. Conclusions We identified specific biological processes, molecules and cell signaling that can be targeted by selected drugs with consequent beneficial effects for cAMP-dependent CHDs. Novelty and Significance What is Known? Congenital Heart Defects are the most frequent heart birth defects including septal defects, hypertrabeculation and non-compacted myocardium. Pde2A hydrolyses the cAMP and cGMP second messengers. Pde2A -deficient mice are embryonic lethal and show cAMP-dependent Congenital Heart Defects. What New Information Does This Article Contribute? We identified several novel pathways altered in hearts of Pde2A -/- embryos. We demonstrated that drugs lowering cAMP levels rescued specific CHDs in Pde2A -/- embryos. We discovered that antioxidants are beneficial for CHDs in Pde2A -/- embryos. The significance of this work relay in molecular discoveries and pharmacological approaches to treat CHDs by using a mouse model that recapitulate the major congenital heart defects. Among the pathways involved in specific defects associated with CHDs, the transcriptome analysis revealed an impairment of genes of the immune system, cardiomyocyte development and contractility, angiogenesis, control of gene transcription and oxidative stress in Pde2A -/- hearts. The scientific community will have open access to the RNA-seq data that can be utilized to further understand the congenital cardiac pathology and clarify the molecular implication in selected defects such as septal and ventricular wall defects. Up to date CHDs, when possible and if identified in time, are mostly treated trough surgery. The identification of drugs blunting the cAMP signaling response or reducing oxidative stress pathways will be useful for setting therapeutic approaches to alleviate CHDs.
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