Lymphoid EVA1 Expression Is Required for DN1-DN3 Thymocytes Transition

Background: Thymus organogenesis and T lymphocyte development are accomplished together during fetal life. Proper development and maintenance of thymus architecture depend on signals generated by a sustained crosstalk between developing thymocytes and stromal elements. Any maturation impairment occurring in either cellular component leads to an aberrant thymic development. Gene expression occurring during T lymphocyte differentiation must be coordinated in a spatio-temporal fashion; one way in which this is achieved is through the regulation by cell-cell adhesion and interactions. Principal Findings: We examined the role played by Epithelial V-like Antigen 1 (EVA1), an Ig adhesion molecule expressed on thymus epithelial cells (TEC) and immature thymocytes, in T cell development by employing RNA interference in vitro and in vivo models. Fetal liver derived haematopoietic progenitors depleted of Eva1, displayed a delayed DN1-DN3 transition and failed to generate CD4CD8 double positive T cells in OP9-DL1 coculture system. In addition, we could observe a coordinated Eva1 up-regulation in stromal and haematopoietic cells in coculture control experiments, suggesting a possible EVA1 involvement in TEC-haematopoietic cells crosstalk mechanisms. Similarly, Rag2-cc double knock out mice, transplanted with Eva1 depleted haematopoietic progenitors displayed a 10-fold reduction in thymus reconstitution and a time delayed thymocytes maturation compared to controls. Conclusions: Our findings show that modulation of Eva1 expression in thymocytes is crucial for lymphocyte physiological developmental progression and stromal differentiation

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

Paracrine signaling from breast cancer cells causes activation of ID4 expression in tumor-associated macrophages

Background: Tumor-associated macrophages (TAMs) constitute a major portion of the leukocyte infiltrate found in breast cancer (BC). BC cells may reprogram TAMs in a pro-angiogenic and immunosuppressive sense. We previously showed that high expression of the ID4 protein in triple-negative BC cells leads to the induction of a proangiogenic program in TAMs also through the downregulation of miR-107. Here, we investigated the expression and function of the ID4 protein in TAMs. Methods: Human macrophages obtained from peripheral blood-derived monocytes (PBDM) and mouse RAW264.7 cells were used as macrophage experimental systems. ID4-correlated mRNAs of the TCGA and E-GEOD-18295 datasets were analyzed. Results: We observed that BC cells determine a paracrine induction of ID4 expression and activation of the ID4 promoter in neighboring macrophages. Interestingly, ID4 expression is higher in macrophages associated with invasive tumor cells compared to general TAMs, and ID4-correlated mRNAs are involved in various pathways that were previously reported as relevant for TAM functions. Selective depletion of ID4 expression in macrophages enabled validation of the ability of ID4 to control the expression of YAP1 and of its downstream targets CTGF and CYR61. Conclusion: Collectively, our results show that activation of ID4 expression in TAMs is observed as a consequence of BC cell paracrine activity and could participate in macrophage reprogramming in BC

Expression of ID4 protein in breast cancer cells induces reprogramming of tumour-associated macrophages

Background: As crucial regulators of the immune response against pathogens, macrophages have been extensively shown also to be important players in several diseases, including cancer. Specifically, breast cancer macrophages tightly control the angiogenic switch and progression to malignancy. ID4, a member of the ID (inhibitors of differentiation) family of proteins, is associated with a stem-like phenotype and poor prognosis in basal-like breast cancer. Moreover, ID4 favours angiogenesis by enhancing the expression of pro-angiogenic cytokines interleukin-8, CXCL1 and vascular endothelial growth factor. In the present study, we investigated whether ID4 protein exerts its pro-angiogenic function while also modulating the activity of tumour-associated macrophages in breast cancer. Methods: We performed IHC analysis of ID4 protein and macrophage marker CD68 in a triple-negative breast cancer series. Next, we used cell migration assays to evaluate the effect of ID4 expression modulation in breast cancer cells on the motility of co-cultured macrophages. The analysis of breast cancer gene expression data repositories allowed us to evaluate the ability of ID4 to predict survival in subsets of tumours showing high or low macrophage infiltration. By culturing macrophages in conditioned media obtained from breast cancer cells in which ID4 expression was modulated by overexpression or depletion, we identified changes in the expression of ID4-dependent angiogenesis-related transcripts and microRNAs (miRNAs, miRs) in macrophages by RT-qPCR. Results: We determined that ID4 and macrophage marker CD68 protein expression were significantly associated in a series of triple-negative breast tumours. Interestingly, ID4 messenger RNA (mRNA) levels robustly predicted survival, specifically in the subset of tumours showing high macrophage infiltration. In vitro and in vivo migration assays demonstrated that expression of ID4 in breast cancer cells stimulates macrophage motility. At the molecular level, ID4 protein expression in breast cancer cells controls, through paracrine signalling, the activation of an angiogenic programme in macrophages. This programme includes both the increase of angiogenesis-related mRNAs and the decrease of members of the anti-angiogenic miR-15b/107 group. Intriguingly, these miRNAs control the expression of the cytokine granulin, whose enhanced expression in macrophages confers increased angiogenic potential. Conclusions: These results uncover a key role for ID4 in dictating the behaviour of tumour-associated macrophages in breast cancer

Argonaute 2 as novel molecular determinant for myeloid differentiation

microRNAs (miRNAs) are emerging as crucial factors for the establishment of complex regulatory circuitries involved in the regulation of hematopoietic cell fate determination. These small non-coding RNAs to exert their functional activity are assembled in RNA-induced silencing complexes (RISCs), where a member of Argonaute (Ago) family of proteins plays a central role in miRNA-mRNA target interaction and gene silencing. In human cells the miRNAs-Ago complex can also localize in the nucleus where Ago proteins can associate with promoter gene sequences to impact heterochromatin genomic structure and transcriptional silencing (Janowski BA et al., 2006; Meister G., 2013). By using human myeloid cell lines and acute myeloid leukemia (AML) primary blasts we highlight Ago2 as a new player in myeloid cell fate determination. We observed that: i) Ago2 protein levels are strongly increased during 1,25-dihydroxyvitamin D3 (D3)-induced monocyte differentiation, whereas are down-regulated during Retinoic Acid (RA)-induced granulocyte differentiation; ii) Ago2 depletion by shRNA or small chemical compounds disrupting both miRNA-Ago2 complex interaction and Ago2 chromatin localization, results in a strong improvement of the RA-dependent myeloid differentiation. These results are bringing out that the down-regulation of Ago2 expression/functional activity is required during RA-dependent myeloid differentiation and may represent a molecular determinant for the improvement of RA-treatment response in leukemic myeloid progenitors cells

CRITICAL ROLE OF MICRORNA BIOGENESIS PATHWAY DURING MYELOID CELL FATE DETERMINATION

ABSTRACT Hematopoiesis is a multistage process where a pluripotent self-renewing hematopoietic stem cell (HSC) gives rise to all blood cell lineages. The clonal expansion of hematopoietic precursors blocked at different stages of differentiation characterized acute myeloid leukemia (AML). MicroRNAs (miRNAs) are emerging as constituents of evolutionary highly conserved molecular pathways regulating cell fate decision in several development programs. In particular, in these last years, miRNAs have been involved in the establishment of myeloid differentiation and leukemogenesis. For this reason, it is not surprising that hematological malignancies are characterized by misregulation of either miRNAs expression or protein involved in miRNA biogenesis pathway. In the first part of this project, here we show, that miR26a is downregulated in primary blasts of AML patients and that, during myeloid differentiation of AML cells, it is induced together with a decrease in c-Myc and Ezh2 levels. In addition, increased levels of miR-26a potentiate the antiproliferative effects of 1,25-dihydroxyvitamin D(3) (VitD) and stimulate myeloid differentiation. Moreover, we identified the transcriptional repressor E2F7 as a novel target of miR- 26a. We show that E2F7 significantly promotes cell cycle progression and inhibits monocytic differentiation of AML cells and that the repression of E2F7 by miR-26a contributes to the increased expression of p21CIP1/WAF1 and to the VitD3-induced monocytic differentiation of AML cells. The results of this study identify a novel aspect of VitD3 action in regulating proliferation and differentiation of AML cells through the miR-26a/E2F7/p21CIP1/WAF1 network. In the second part of this project, we show the role of Argonaute 2 (Ago2), a core component of RISC (RNA-induced silencing complex), in human myeloid differentiation. In particular, we observed that Ago2 protein levels are increased during monocyte differentiation of myeloid progenitors, whereas are downregulated during granulocyte differentiation of human leukemic cell lines and freshly isolated blasts from acute promyelocytic leukemia (APL) patients. We found that the Ago2 silencing, lentivirus mediated, alters the correct modulation of transcription factors and microRNAs involved 5 in monocyte cell fate determination, leading to the reduction of VitD-induced monocytic differentiation and subsequently activation by LPS-induced inflammatory response

CHARACTERIZATION OF THE UNFOLDED PROTEIN RESPONSE ROLE IN DIFFERENTIATION THERAPY OF ACUTE MYELOID LEUKEMIAS

Introduction. Acute myeloid leukemia (AML) is caused by the clonal expansion of hematopoietic myeloid precursors blocked at different stages of differentiation. A subtype of AML, acute promyelocytic leukemia (APL), is a paradigm of differentiation therapy since all-transretinoic acid (ATRA)-based treatments are able to induce leukemic blast terminal differentiation, leading to clinical remission in the majority of APL patients. However, ATRA can lead to systemic toxicity and relapses after initial remission followed by resistance. Furthermore APL accounts for about 10-15% of AML cases and non-APL AML respond only very slightly to ATRA. Thus the search for a strategy to further sensitize AML cells to ATRA is highly needed. ATRA induces differentiation of APL blasts to granulocytes that are secretory cells since they are characterized by the presence of secretory granules containing peptides indispensable for their role in the immune response. The majority of proteins secreted or reside

microRNA biogenesis pathway as therapeutic target for human disease and cancer

The deregulation of miRNAs expression and activity is frequently observed in a wide variety of human pathologies including cancer. Accordingly, growing evidence indicates that the targeting of microRNAs biogenesis and pathways is emerging as a central tool for the development of novel RNA-based drugs and therapies to defeat diseases in humans. In this review we describe the various strategies that can be used to target the microRNAs and specific RNA-binding proteins, involved in the regulation of their production, localization, stability and activity, in human cancer and cardiovascular diseases. We also focus on the efforts that are currently made to enhance the potency and stability of these therapeutic agents and their delivery to modulate in vivo microRNAs pathways. Finally, we present structural data on proteins that belong to the microRNA pathway for small molecules-based target therapy design

Analysis of <i>Eva1</i> expression.

<p><i>Eva1</i> real-time RT-PCR in fetal thymi (A), adult DN subpopulations (B, left panel) and thymocytes from mutant mice (B, right panel). mRNA from flow cytometrically purified TECs (CD45-) and intrathymic haematopoietic cells (CD45+) or DN1-3 subpopulations was reverse transcribed and used as the template for PCR with <i>Eva1</i>-specific primers. All samples were normalized to the geometric mean of the GAPDH housekeeping gene. NB, newborn; 2mth, two months; WT, wild type; ΔCAM, Tg-Calcineurin; <i>Prkdc</i>^scid, protein kinase, DNA-activated, catalytic polypeptide (C, upper panel) Eva1 interference in LSK cells by lentiviral vector was controlled by real time RT-PCR. LSK-CT cells shown a comparable expression of <i>Eva1</i> while LSK-EVAi cells shown a drastic decrease of <i>Eva1</i> expression, indicating that the interference has occurred. (C, lower panel), fluorescence microscopy analysis confirming <i>Eva1</i> interference in LSK-EVAi cells. LSK-WT, uninfected LSK; LSK-CT, LSK infected with a non-interfering lentiviral vector; LSK-EVAi, LSK infected with a <i>Eva1</i>-interfering lentiviral vector.</p