Regulation of ribosome function by RNA modifications in hematopoietic development and leukemia: it is not only a matter of m6A

Growth and maturation of hematopoietic stem cells (HSCs) are largely controlled at both transcriptional and post-transcriptional levels. In particular, hematopoietic development requires a tight control of protein synthesis. Furthermore, translational deregulation strongly contributes to hematopoietic malignancies. Researchers have recently identified a new layer of gene expression regulation that consists of chemical modification of RNA species, which led to the birth of the epitranscriptomics field. RNA modifications provide an additional level of control in hematopoietic development by acting as post-transcriptional regulators of lineage-specific genetic programs. Other reviews have already described the important role of the N6-methylation of adenosine (m6A) within mRNA species in regulating hematopoietic differentiation and diseases. The aim of this review is to summarize the current status of the role of RNA modifications in the regulation of ribosome function, beyond m6A. In particular, we discuss the importance of RNA modifications in tRNA and rRNA molecules. By balancing translational rate and fidelity, they play an important role in regulating normal and malignant hematopoietic development

Not only P-glycoprotein: amplification of the ABCB1-containing chromosome region 7q21 confers multidrug resistance upon cancer cells by coordinated overexpression of an assortment of resistance-related proteins

The development of drug resistance continues to be a dominant hindrance toward curative cancer treatment. Overexpression of a wide-spectrum of ATP-dependent efflux pumps, and in particular of ABCB1 (P-glycoprotein or MDR1) is a well-known resistance mechanism for a plethora of cancer chemotherapeutics including for example taxenes, anthracyclines, Vinca alkaloids, and epipodopyllotoxins, demonstrated by a large array of published papers, both in tumor cell lines and in a variety of tumors, including various solid tumors and hematological malignancies. Upon repeated or even single dose treatment of cultured tumor cells or tumors in vivo with anti-tumor agents such as paclitaxel and doxorubicin, increased ABCB1 copy number has been demonstrated, resulting from chromosomal amplification events at 7q11.2-21 locus, leading to marked P-glycoprotein overexpression, and multidrug resistance (MDR). Clearly however, additional mechanisms such as single nucleotide polymorphisms (SNPs) and epigenetic modifications have shown a role in the overexpression of ABCB1 and of other MDR efflux pumps. However, notwithstanding the design of 4 generations of ABCB1 inhibitors and the wealth of information on the biochemistry and substrate specificity of ABC transporters, translation of this vast knowledge from the bench to the bedside has proven to be unexpectedly difficult. Many studies show that upon repeated treatment schedules of cell cultures or tumors with taxenes and anthracyclines as well as other chemotherapeutic drugs, amplification, and/or overexpression of a series of genes genomically surrounding the ABCB1 locus, is observed. Consequently, altered levels of other proteins may contribute to the establishment of the MDR phenotype, and lead to poor clinical outcome. Thus, the genes contained in this ABCB1 amplicon including ABCB4, SRI, DBF4, TMEM243, and RUNDC3B are overexpressed in many cancers, and especially in MDR tumors, while TP53TG1 and DMTF1 are bona fide tumor suppressors. This review describes the role of these genes in cancer and especially in the acquisition of MDR, elucidates possible connections in transcriptional regulation (co-amplification/repression) of genes belonging to the same ABCB1 amplicon region, and delineates their novel emerging contributions to tumor biology and possible strategies to overcome cancer MDR

MicroRNA pathways as new functional axes in cell fate determination

The identification of a series of non-coding RNA (ncRNA) families, with regulatory roles in several biological processes, represents one of the most relevant advances derived from transcriptome analysis. These ncRNAs are actively transcribed from the genome of many organisms. Among them microRNAs (miRNAs) are small regulatory, single-stranded, RNA molecules (19–25 nucleotides in length) that are generated in the nucleus as hairpin primary transcripts. miRNAs processing is carried out initially in the nucleus and later on in the cytoplasm, where the mature miRNAs molecules, through a limited base-pairing complementarity, destabilize or block the translation of their phylogenetically conserved target transcripts [1]. miRNAs exert their function assembled in the RNA-induced silencing complexes (RISCs), where members of Argonaute (Ago) family of proteins provide a unique platform for target recognition and gene silencing. Interestingly, recent evidences also suggest that miRNAs and epigenetic pathways appear to form a complex regulatory circuit that modulates the expression of an increasing number of genes in the genome. miRNAs are not only modulated by epigenetic regulation but it is now emerging that miRNAs also have specific epigenetic functions for the regulation of the transcriptional landscape of the cell. Following the identification of a functional miRNAs pathway in C. elegans, their contribution to both embryonic development and maintenance of tissue homeostasis in adults has rapidly emerged also in mammals. miRNAs exhibit a developmental stage- and tissue-specific expression and are present in critical regulatory pathways to regulate stem cells function, cell lineage specification/differentiation, maintenance of cell identity and tissue induction, growth and morphogenesis. The identification of the molecular pathways involving miRNAs and specific RNA-binding proteins, responsible for the regulation of their production, localization, stability and activity, is gaining crucial importance for the characterization of physiological and pathological processes of human development

Retinoic acid-induced differentiation sensitizes myeloid progenitors cells to ER stress

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 miR-223 host non-coding transcript linc-223 induces IRF4 expression in acute myeloid leukemia by acting as a competing endogenous RNA

Alterations in genetic programs required for terminal myeloid differentiation and aberrant proliferation characterize acute myeloid leukemia (AML) cells. Here, we identify the host transcript of miR-223, linc-223, as a novel functional long non-coding RNA (lncRNA) in AML. We show that from the primary nuclear transcript, the alternative production of miR-223 and linc-223 is finely regulated during monocytic differentiation. Moreover, linc-223 expression inhibits cell cycle progression and promotes monocytic differentiation of AML cells. We also demonstrate that endogenous linc-223 localizes in the cytoplasm and acts as a competing endogenous RNA for miR-125-5p, an oncogenic microRNA in leukemia. In particular, we show that linc-223 directly binds to miR-125-5p and that its knockdown increases the repressing activity of miR-125-5p resulting in the downregulation of its target interferon regulatory factor 4 (IRF4), which it was previously shown to inhibit the oncogenic activity of miR-125-5p in vivo. Furthermore, data from primary AML samples show significant downregulation of linc-223 in different AML subtypes. Therein, these findings indicate that the newly identified lncRNA linc-223 may have an important role in myeloid differentiation and leukemogenesis, at least in part, by cross-talking with IRF4 mRNA

C/EBPα-p30 protein induces expression of the oncogenic long non-coding RNA UCA1 in acute myeloid leukemia

Accumulating evidences indicate that different long non-coding RNAs (lncRNAs) might play a relevant role in tumorigenesis, with their expression and function already associated to cancer development and progression. CCAAT/enhancer-binding protein-α (CEBPA) is a critical regulator of myeloid differentiation whose inactivation contributes to the development of acute myeloid leukemia (AML). Mutations in C/EBPα occur in around 10% of AML cases, leading to the expression of a 30-kDa dominant negative isoform (C/EBPα-p30). In this study, we identified the oncogenic urothelial carcinoma associated 1 (UCA1) lncRNA as a novel target of the C/EBPα-p30. We show that wild-type C/EBPα and C/EBPα-p30 isoform can bind the UCA1 promoter but have opposite effects on UCA1 expression. While wild-type C/EBPα represses, C/EBPα-p30 can induce UCA1 transcription. Notably, we also show that UCA1 expression increases in cytogenetically normal AML cases carrying biallelic CEBPA mutations. Furthermore, we demonstrate that UCA1 sustains proliferation of AML cells by repressing the expression of the cell cycle regulator p27kip1. Thus, we identified, for the first time, an oncogenic lncRNA functioning in concert with the dominant negative isoform of C/EBPα in AML

METTL3 regulates WTAP protein homeostasis

The Wilms tumor 1 (WT1)-associated protein (WTAP) is upregulated in many tumors, including, acute myeloid leukemia (AML), where it plays an oncogenic role by interacting with different proteins involved in RNA processing and cell proliferation. In addition, WTAP is also a regulator of the nuclear complex required for the deposition of N6-methyladenosine (m6A) into mRNAs, containing the METTL3 methyltransferase. However, it is not clear if WTAP may have m6A-independent regulatory functions that might contribute to its oncogenic role. Here, we show that both knockdown and overexpression of METTL3 protein results in WTAP protein upregulation, indicating that METTL3 levels are critical for WTAP protein homeostasis. However, we show that WTAP upregulation is not sufficient to promote cell proliferation in the absence of a functional METTL3. Therein, these data indicate that the reported oncogenic function of WTAP is strictly connected to a functional m6A methylation complex

Oncogenic microRNAs characterization in clear cell renal cell carcinoma

A key challenge for the improvement of clear cell renal cell carcinoma (ccRCC) management could derive from a deeper characterization of the biology of these neoplasms that could greatly improve the diagnosis, prognosis and treatment choice. The aim of this study was to identify specific miRNAs that are deregulated in tumor vs. normal kidney tissues and that could impact on the biology of ccRCC. To this end we selected four miRNAs (miR-21-5p, miR-210-3p, miR-185-5p and miR-221-3p) and their expression has been evaluated in a retrospective cohort of formalin-fixed paraffin-embedded (FFPE) tissues from 20 ccRCC patients who underwent surgical nephrectomy resection. miR-21-5p and miR-210-3p resulted the most significantly up-regulated miRNAs in this patient cohort, highlighting these onco-miRNAs as possible relevant players involved in ccRCC tumorigenesis. Thus, this study reports the identification of specific oncogenic miRNAs that are altered in ccRCC tissues and suggests that they might be useful biomarkers in ccRCC management

Secreted miR-210-3p as non-invasive biomarker in clear cell renal cell carcinoma

The most common subtype of renal cell carcinoma (RCC) is clear cell RCC (ccRCC). It accounts for 70-80% of all renal malignancies representing the third most common urological cancer after prostate and bladder cancer. The identification of non-invasive biomarkers for the diagnosis and responsiveness to therapy of ccRCC may represent a relevant step-forward in ccRCC management. The aim of this study is to evaluate whether specific miRNAs deregulated in ccRCC tissues present altered levels also in urine specimens. To this end we first assessed that miR-21-5p, miR-210-3p and miR-221-3p resulted upregulated in ccRCC fresh frozen tissues compared to matched normal counterparts. Next, we evidenced that miR-210-3p resulted significantly upregulated in 38 urine specimens collected from two independent cohorts of ccRCC patients at the time of surgery compared to healthy donors samples. Of note, miR- 210-3p levels resulted significantly reduced in follow-up samples. These results point to miR-210-3p as a potential non-invasive biomarker useful not only for diagnosis but also for the assessment of complete resection or response to treatment in ccRCC management