MicroRNAs 17-5p-20a-106a control monocytopoiesis through AML1 targeting and M-CSF receptor upregulation.

We investigated the role of microRNAs (miRNA) 17-5p, 20a and 106a in monocytic differentiation and maturation. In unilineage monocytic culture generated by haematopoietic progenitor cells these miRNAs are downregulated, whereas the transcription factor acute myeloid leukaemia-1 (AML1; also known as Runt-related transcription factor 1, Runx1) is upregulated at protein but not mRNA level. As miRNAs 17-5p, 20a and 106a bind the AML1 mRNA 3'UTR, their decline may unblock AML1 translation. Accordingly, transfection with miRNA 17-5p-20a-106a suppresses AML1 protein expression, leading to M-CSF receptor (M-CSFR) downregulation, enhanced blast proliferation and inhibition of monocytic differentiation and maturation. Treatment with anti-miRNA 17-5p, 20a and 106a causes opposite effects. Knockdown of AML1 or M-CSFR by short interfering RNA (siRNA) mimics the action of the miRNA 17-5p-20a-106a, confirming that these miRNAs target AML1, which promotes M-CSFR transcription. In addition, AML1 binds the miRNA 17-5p-92 and 106a-92 cluster promoters and transcriptionally inhibits the expression of miRNA 17-5p-20a-106a. These studies indicate that monocytopoiesis is controlled by a circuitry involving sequentially miRNA 17-5p-20a-106a, AML1 and M-CSFR, whereby miRNA 17-5p-20a-106a function as a master gene complex interlinked with AML1 in a mutual negative feedback loop.

MicroRNA-133 controls cardiac hypertrophy.

Growing evidence indicates that microRNAs (miRNAs or miRs) are involved in basic cell functions and oncogenesis. Here we report that miR-133 has a critical role in determining cardiomyocyte hypertrophy. We observed decreased expression of both miR-133 and miR-1, which belong to the same transcriptional unit, in mouse and human models of cardiac hypertrophy. In vitro overexpression of miR-133 or miR-1 inhibited cardiac hypertrophy. In contrast, suppression of miR-133 by 'decoy' sequences induced hypertrophy, which was more pronounced than that after stimulation with conventional inducers of hypertrophy. In vivo inhibition of miR-133 by a single infusion of an antagomir caused marked and sustained cardiac hypertrophy. We identified specific targets of miR-133: RhoA, a GDP-GTP exchange protein regulating cardiac hypertrophy; Cdc42, a signal transduction kinase implicated in hypertrophy; and Nelf-A/WHSC2, a nuclear factor involved in cardiogenesis. Our data show that miR-133, and possibly miR-1, are key regulators of cardiac hypertrophy, suggesting their therapeutic application in heart disease.

Identification and expansion of human colon-cancer-initiating cells.

Colon carcinoma is the second most common cause of death from cancer. The isolation and characterization of tumorigenic colon cancer cells may help to devise novel diagnostic and therapeutic procedures. Although there is increasing evidence that a rare population of undifferentiated cells is responsible for tumour formation and maintenance, this has not been explored for colorectal cancer. Here, we show that tumorigenic cells in colon cancer are included in the high-density CD133+ population, which accounts for about 2.5% of the tumour cells. Subcutaneous injection of colon cancer CD133+ cells readily reproduced the original tumour in immunodeficient mice, whereas CD133- cells did not form tumours. Such tumours were serially transplanted for several generations, in each of which we observed progressively faster tumour growth without significant phenotypic alterations. Unlike CD133- cells, CD133+ colon cancer cells grew exponentially for more than one year in vitro as undifferentiated tumour spheres in serum-free medium, maintaining the ability to engraft and reproduce the same morphological and antigenic pattern of the original tumour. We conclude that colorectal cancer is created and propagated by a small number of undifferentiated tumorigenic CD133+ cells, which should therefore be the target of future therapies.

NFI-A directs the fate of hematopoietic progenitors to the erythroid or granulocytic lineage and controls beta-globin and G-CSF receptor expression.

It is generally conceded that selective combinations of transcription factors determine hematopoietic lineage commitment and differentiation. Here we show that in normal human hematopoiesis the transcription factor nuclear factor I-A (NFI-A) exhibits a marked lineage-specific expression pattern: it is upmodulated in the erythroid (E) lineage while fully suppressed in the granulopoietic (G) series. In unilineage E culture of hematopoietic progenitor cells (HPCs), NFI-A overexpression or knockdown accelerates or blocks erythropoiesis, respectively: notably, NFI-A overexpression restores E differentiation in the presence of low or minimal erythropoietin stimulus. Conversely, NFI-A ectopic expression in unilineage G culture induces a sharp inhibition of granulopoiesis. Finally, in bilineage E + G culture, NFI-A overexpression or suppression drives HPCs into the E or G differentiation pathways, respectively. These NFI-A actions are mediated, at least in part, by a dual and opposite transcriptional action: direct binding and activation or repression of the promoters of the beta-globin and G-CSF receptor gene, respectively. Altogether, these results indicate that, in early hematopoiesis, the NFI-A expression level acts as a novel factor channeling HPCs into either the E or G lineage.

Correlations between progression of coronary artery disease and circulating endothelial progenitor cells.

The pathophysiology of coronary artery disease (CAD) progression is not well understood. Endothelial progenitor cells (EPCs) may have an important role. In the present observational cohort study we assessed the number of circulating EPCs in 136 patients undergoing elective percutaneous coronary intervention and who had at least one major epicardial vessel with a nonsignificant stenosis [<50% diameter stenosis (DS)], and the relationship between plasma EPC levels and the 24-mo progression of the nonsignificant coronary artery lesion. The following cell populations were analyzed: CD34(+), CD133(+), CD34(+)/KDR(+), CD34(+)/VE cadherin(+), and endothelial cell colony-forming units (CFU-ECs). Progression was defined as a >15% DS increase of the objective vessel at follow-up. At 24 mo, 57 patients (42%) experienced significant progression. Independent predictors of disease progression were LDL cholesterol > 100 mg/dl (OR=1.03; 95% CI 1.01-1.04; P=0.001), low plasma levels of CFU-ECs (OR=3.99; 95% CI 1.54-10.37; P=0.005), and male sex (OR=3.42; 95% CI 1.15-10.22; P=0.027). Circulating levels of EPCs are significantly lower in patients with angiographic CAD progression.

Absence of caspase 8 and high expression of PED protect primitive neural cells from cell death.

The mechanisms that control neural stem and progenitor cell survival are unknown. In several pathological conditions, death receptor (DR) ligands and inflammatory cytokines exert a deleterious effect on neurons, whereas primitive neural cells migrate and survive in the site of lesion. Here, we show that even in the presence of inflammatory cytokines, DRs are unable to generate death signals in primitive neural cells. Neural stem and progenitor cells did not express caspase 8, the presence of which is required for initiating the caspase cascade. However, exogenous or cytokine-mediated expression of caspase 8 was not sufficient to restore their DR sensitivity. Searching for molecules potentially able to block DR death-inducing signaling complex (DISC), we found that primitive neural cells expressed high levels of the death effector domain-containing protein PED (also known as PEA-15). PED localized in the DISC and prevented caspase 8 recruitment and activation. Moreover, lentiviral-mediated delivery of PED antisense DNA resulted in dramatic down-regulation of the endogenous gene expression and sensitization of primitive neural cells to apoptosis mediated by inflammatory cytokines and DRs. Thus, absence of caspase 8 and high expression of PED constitute two levels of protection from apoptosis induced by DRs and inflammatory cytokines in neural stem and progenitor cells.

Mechanism of human Hb switching: a possible role of the kit receptor/miR 221-222 complex.

BACKGROUND: The human hemoglobin switch (HbF-->HbA) takes place in the peri/post-natal period. In adult life, however, the residual HbF (<1%) may be partially reactivated by chemical inducers and/or cytokines such as the kit ligand (KL). MicroRNAs (miRs) play a pivotal role in normal hematopoiesis: downmodulation of miR-221/222 stimulates human erythropoietic proliferation through upmodulation of the kit receptor. DESIGN AND METHODS: We have explored the possible role of kit/KL in perinatal Hb switching by evaluating: i) the expression levels of both kit and kit ligand on CD34(+) cells and in plasma isolated from pre-, mid- and full-term cord blood samples; ii) the reactivation of HbF synthesis in KL-treated unilineage erythroid cell cultures; iii) the functional role of miR-221/222 in HbF production. RESULTS: In perinatal life, kit expression showed a gradual decline directly correlated to the decrease of HbF (from 80-90% to <30%). Moreover, in full-term cord blood erythroid cultures, kit ligand induced a marked increase of HbF (up to 80%) specifically abrogated by addition of the kit inhibitor imatinib, thus reversing the Hb switch. MiR-221/222 expression exhibited rising levels during peri/post-natal development. In functional studies, overexpression of these miRs in cord blood progenitors caused a remarkable decrease in kit expression, erythroblast proliferation and HbF content, whereas their suppression induced opposite effects. CONCLUSIONS: Our studies indicate that human perinatal Hb switching is under control of the kit receptor/miR 221-222 complex. We do not exclude, however, that other mechanisms (i.e. glucocorticoids and the HbF inhibitor BCL11A) may also contribute to the peri/post-natal Hb switch.

MicroRNA 223-dependent expression of LMO2 regulates normal erythropoiesis.

BACKGROUND: MicroRNAs are small non-coding RNAs that regulate gene expression through mRNA degradation or translational inhibition. MicroRNAs are emerging as key regulators of normal hematopoiesis and hematologic malignancies. Several miRNAs are differentially expressed during hematopoiesis and their specific expression regulates key functional proteins involved in hematopoietic lineage differentiation. This study focused on the functional role of microRNA-223 (miR-223) on erythroid differentiation. DESIGN AND METHODS: Purified cord blood CD34+ hematopoietic progenitor cells were grown in strictly controlled conditions in the presence of saturating dosage of erythropoietin to selectively induce erythroid differentiation. The effects of enforced expression of miR-223 in unilin-eage erythroid cultures were evaluated in liquid phase culture experiments and clonogenic studies. RESULTS: In unilineage erythroid culture of cord blood CD34+ hematopoietic progenitor cells miR-223 is down-regulated, whereas LMO2, an essential protein for erythroid differentiation, is up-regulated. Functional studies showed that enforced expression of miR-223 reduces the mRNA and protein levels of LMO2, by binding to LMO2 3' UTR, and impairs differentiation of erythroid cells. Accordingly, knockdown of LMO2 by short interfering RNA mimics the action of miR-223. Furthermore, hematopoietic progenitor cells transduced with miR-223 showed a significant reduction of their erythroid clonogenic capacity, suggesting that downmodulation of this miRNA is required for erythroid progenitor recruitment and commitment. CONCLUSIONS: These results show that the decline of miR-223 is an important event for erythroid differentiation that leads to the expansion of erythroblast cells at least partially mediated by unblocking LMO2 protein expression.

Chemotherapy-induced thrombocytopenia derives from the selective death of megakaryocyte progenitors and can be rescued by stem cell factor.

Thrombocytopenia is a common side effect of chemotherapy, responsible for increased risk of bleeding and delay of treatment schedules in cancer patients. It is currently unknown how chemotherapeutic agents affect platelet production and whether the platelet precursors megakaryocytes represent a direct target of cytotoxic drugs. We investigated the effects of chemotherapeutic agents on primary megakaryocytes by using a culture system that recapitulates in vitro human megakaryopoiesis and found that cytotoxic drugs predominantly destroyed megakaryocytic progenitors at early stages of differentiation. Immature megakaryocytes could be protected from chemotherapeutic agents by the cytokine stem cell factor (SCF), which binds the c-kit receptor expressed on hematopoietic stem and progenitor cells. In chemotherapy-treated megakaryocytes, SCF activated Akt, neutralized the mitochondrial apoptotic machinery, and inhibited caspase activity. Interfering with Akt activation abrogated the antiapoptotic effects of SCF, whereas exogenous expression of constitutively active Akt inhibited drug-induced apoptosis of primary megakaryocytes, indicating the Akt pathway as primarily responsible for SCF-mediated protection of megakaryocyte progenitors. These results indicate apoptosis of megakaryocyte progenitors as a major cause of chemotherapy-induced thrombocytopenia and suggest that SCF may be used to prevent platelet loss in cancer patients with c-kit-negative tumors.

Isolation and characterization of CD146+ multipotent mesenchymal stromal cells.

Mesenchymal stromal cells (MSCs) represent a bone marrow (BM) population, classically defined by five functional properties: extensive proliferation, ability to differentiate into osteoblasts, chondrocytes, adipocytes, and stromal cells-supporting hematopoiesis. However, research progress in this area has been hampered by lack of suitable markers and standardized procedures for MSC isolation. We have isolated a CD146(+) multipotent MSC population from 20 human BM donors displaying the phenotype of self-renewing osteoprogenitors; an extensive 12-week proliferation; and the ability to differentiate in osteoblasts, chondrocytes, adipocytes, and stromal cells supporting hematopoiesis. Furthermore, the CD146(+) MSCs secrete a complex combination of growth factors (GFs) controlling hematopoietic stem cells (HSCs) function, while providing a >2-log increase in the long-term culture (LTC) colony output in 8-week LTC over conventional assays. The hematopoietic stromal function exhibited by the MSCs was further characterized by manipulating LTCs with the chemical inhibitors Imatinib or SU-5416, targeting two GF receptors (GFRs), KIT or VEGFR2/1, respectively. Both treatments similarly impaired LTC colony output, indicating key roles for these two GF/GFR interactions to support LTC-initiating cell activity. CD146(+) MSCs may thus represent a tool to explore the MSC-HSC cross-talk in an in vitro surrogate model for HSC "niches," and for regenerative therapy studies. In addition, the MSC microRNA (miRNA) expression profile was analyzed by microarrays in both basic conditions and chondrogenic differentiation. Our analysis revealed that several miRNAs are modulated during chondrogenesis, and many of their putative targets are genes involved in chondrogenic differentiation.

Methylation damage response in hematopoietic progenitor cells.

The cellular response to methylation DNA damage was compared in multipotent CD34(+) hematopoietic stem cells and mature CD34(-) cells isolated from cord blood of the same donor. Cytofluorimetric analysis of freshly isolated cord blood cells indicated that both cell types were in the G0/G1 phase of the cell cycle. Quantitative RT-PCR identified a general trend towards high expression of several DNA repair genes in CD34(+) cells compared to their terminally differentiated CD34(-) counterparts. The overexpressed genes included members of the mismatch repair (MMR) (MSH2, MSH6, MLH1, PMS2), base excision repair (AAG, APEX), DNA damage reversal (O(6)-methylguanine DNA methyltransferase) (MGMT), and DNA double strand breaks repair pathways. These differences in gene expression were not apparent in CD34(+) and CD34(-) cells obtained following expansion of CD34(+) cells in a medium containing early acting cytokines. Early progenitor CD34(+) and early precursor CD34(-) cells form the two populations isolated under these experimental conditions, and both contain a significant proportion of cycling cells. The methylating agent N-methyl-N-nitrosourea (MNU) induced similar levels of apoptosis in these cycling CD34(+) and CD34(-) cells. Cytotoxicity required the presence of the MGMT inhibitor O(6)-benzylguanine and the timing of MNU cell death (48 and 72h) was similar in CD34(+) and CD34(-) cells. These data indicate that cycling CD34(+) and CD34(-) cells are equally sensitive to methylation damage. MGMT provides significant protection against MNU toxicity and MGMT and MMR play the expected roles in the MNU sensitivity of these cells.

MicroRNA 155 modulates megakaryopoiesis at progenitor and precursor level by targeting Ets-1 and Meis1 transcription factors.

MicroRNAs (miRNAs) control basic biological functions and are emerging as key regulators of haematopoiesis. This study focused on the functional role of MIRN155 on megakaryocytic (MK) differentiation of human cord blood CD34+ haematopoietic progenitor cells (HPCs). MIRN155, abundantly expressed in early HPCs, decreases sharply during MK differentiation. Functional studies showed that enforced expression of MIRN155 impairs proliferation and differentiation of MK cells. Furthermore, HPCs transfected with MIRN155 showed a significant reduction of their MK clonogenic capacity, suggesting that down-modulation of this miRNA favours MK progenitor differentiation. Consistent with this observation, MIRN155 downregulates, by directly binding to their 3'-UTR, the expression of Ets-1 and Meis1, two transcription factors with well-known functions in MK cells. These results show that the decline of MIRN155 is required for MK proliferation and differentiation at progenitors and precursors level and indicate that sustained expression of MIRN155 inhibits megakaryopoiesis.

Role of microRNAs in haemopoiesis, heart hypertrophy and cancer.

miRNAs (microRNAs) are important regulatory molecules that control gene expression in all eukaryotes. miRNAs play an essential role in basic cellular activities such as proliferation, differentiation, morphogenesis and apoptosis. In haemopoiesis, several miRNA-based pathways have been identified. Importantly, miRNA mutations or mis-expression correlate with various human diseases. In cancer, deregulated miRNAs can function as tumour suppressors or oncogenes. The present review focuses on the recent literature concerning the role of miRNAs in three different research areas: haematology, cardiology and oncology, with particular focus on the results obtained by our group.

Resistance of acute myeloid leukemic cells to the triterpenoid CDDO-Imidazolide is associated with low caspase-8 and FADD levels.

The synthetic triterpenoid CDDO-Im-induced apoptosis of patient-derived AML blasts: 11/25 AMLs were highly sensitive, while the remaining were moderately sensitive to CDDO-Im. The addition of TRAIL significantly potentiated the cytotoxic effect of CDDO-Im, through mechanisms involving the induction of TRAIL-R1/TRAIL-R2 and downmodulation of TRAIL-R3/TRAIL-R4. Biochemical studies showed that CDDO-Im: induced a rapid and marked GSH depletion and antioxidants (GSH or NAC) completely inhibited its pro-apoptotic effect; sequentially activated caspase-8, -9 and -3; caspase inhibitors partially protected AML blasts from CDDO-Im-induced apoptosis; resistance of AML blasts to CDDO-Im-induced apoptosis correlated with low caspase-8/FADD and high Bcl-X(L) expression in leukemic blasts.

Role of microRNAs in drug-resistant ovarian cancer cells.

OBJECTIVES: Chemotherapy is the preferred therapeutic approach for the therapy of advanced ovarian cancer, but a successful long-term treatment is prevented by the development of drug resistance. Recent works have underlined the involvement of non-coding RNAs, microRNAs (miRNAs) in cancer development, with several conjectures regarding their possible involvement in the evolution of drug resistance. This work was aimed to identify selected microRNAs involved in the development of chemoresistance in ovarian cancer. METHODS: High-throughput analysis of the miRNA profile in a panel of paclitaxel- (A2780TAX, A2780TC1 and A2780TC3) and cisplatin-resistant (A2780CIS) cells was assessed using a microarray platform and subsequent validation with qPCR and Northern blots. Downstream target validation was performed for miR-130a and the target M-CSF.] RESULTS: Six miRNAs (let-7e, miR-30c, miR-125b, miR-130a and miR-335) were always diversely expressed in all the resistant cell lines. Let-7e was upregulated in A2780TAX cells, while it was downregulated in the other resistant cell lines. The opposite phenomenon was obtained for miR-125b, which was downregulated in A2780TAX and upregulated in the other cell lines. The miR-30c, miR-130a and miR-335 were downregulated in all the resistant cell lines, thereby suggesting a direct involvement in the development of chemoresistance. Finally downstream target validation was proven for the miR-130a, whose downregulation was linked to the translational activation of the M-CSF gene, a known resistance factor for ovarian cancer. CONCLUSIONS: Our results indicate that ovarian cancer drug resistance is associated with a distinct miRNA fingerprint, and miRNA microarrays could represent a prognostic tool to monitor the chemotherapy outcome.

Glycated fibroblast growth factor-2 is quickly produced in vitro upon low-millimolar glucose treatment and detected in vivo in diabetic mice.

Angiogenesis impairment in hyperglycemic patients represents a leading cause of severe vascular complications of both type-1 and -2 diabetes mellitus (DM). Angiogenesis dysfunction in DM is related to glycemic control; however, molecular mechanisms involved are still unclear. Fibroblast growth factor-2 (FGF-2) is a potent angiogenic factor and, according to previous evidence, may represent a key target of molecular modifications triggered by high-sugar exposure. Therefore, the purpose of this study was to investigate whether short incubation with hyperglycemic levels of glucose affected FGF-2 and whether glucose-modified FGF-2 was detectable in vivo. Biochemical analyses carried out with SDS-PAGE, fluorescence emission, mass-spectrometry, immunoblot, and competitive ELISA experiments demonstrated that human FGF-2 undergoes a rapid and specific glycation upon 12.5-50 mm glucose exposure. In addition, FGF-2 exposed for 30 min to 12.5 mm glucose lost mitogenic and chemotactic activity in a time- and dose-dependent manner. Under similar conditions, binding affinity to FGF receptor 1 was dramatically reduced by 20-fold, as well as FGF receptor 1 and ERK-1/2 phosphorylation, and FGF-2 lost about 45% of angiogenic activity in two different in vivo angiogenic (Matrigel and chorioallantoic-membrane) assays. Such glucose-induced modification was specific, because other angiogenic growth factors, namely platelet-derived growth factor BB and placental-derived growth factor were not significantly or markedly less modified. Finally, for the first time, glycated-FGF-2 was detected in vivo, in tissues from hyperglycemic nonobese diabetic mice, in significantly higher amounts than in normoglycemic mice. In conclusion, hyperglycemic levels of glucose may strongly affect FGF-2 structure and impair its angiogenic features, and endogenous glycated-FGF-2 is present in diabetic mice, indicating a novel pathogenetic mechanism underlying angiogenesis defects in DM.

Inhibition of DNA methylation sensitizes glioblastoma for tumor necrosis factor-related apoptosis-inducing ligand-mediated destruction.

Life expectancy of patients affected by glioblastoma multiforme is extremely low. The therapeutic use of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been proposed to treat this disease based on its ability to kill glioma cell lines in vitro and in vivo. Here, we show that, differently from glioma cell lines, glioblastoma multiforme tumors were resistant to TRAIL stimulation because they expressed low levels of caspase-8 and high levels of the death receptor inhibitor PED/PEA-15. Inhibition of methyltransferases by decitabine resulted in considerable up-regulation of TRAIL receptor-1 and caspase-8, down-regulation of PED/PEA-15, inhibition of cell growth, and sensitization of primary glioblastoma cells to TRAIL-induced apoptosis. Exogenous caspase-8 expression was the main event able to restore TRAIL sensitivity in primary glioblastoma cells. The antitumor activity of decitabine and TRAIL was confirmed in vivo in a mouse model of glioblastoma multiforme. Evaluation of tumor size, apoptosis, and caspase activation in nude mouse glioblastoma multiforme xenografts showed dramatic synergy of decitabine and TRAIL in the treatment of glioblastoma, whereas the single agents were scarcely effective in terms of reduction of tumor mass, apoptosis induction, and caspase activation. Thus, the combination of TRAIL and demethylating agents may provide a key tool to overcome glioblastoma resistance to therapeutic treatments.