Marina Petrini

miR-126&126* Restored Expressions Play a Tumor Suppressor Role by Directly Regulating ADAM9 and MMP7 in Melanoma

The abnormal expression of several microRNAs has a causal role in tumorigenesis with either antineoplastic or oncogenic functions. Here we demonstrated that miR-126 and miR-126* play a tumor suppressor role in human melanoma through the direct or indirect repression of several key oncogenic molecules. The expression levels of miR-126&126* were elevated in normal melanocytes and primary melanoma cell lines, whereas they markedly declined in metastatic cells. Indeed, the restored expression of miR-126&126* in two advanced melanoma cell lines was accompanied by a significant reduction of proliferation, invasion and chemotaxis in vitro as well as of growth and dissemination in vivo. In accordance, the reverse functional effects were obtained by knocking down miR-126&126* by transfecting antisense LNA oligonucleotides in melanoma cells. Looking for the effectors of these antineoplastic functions, we identified ADAM9 and MMP7, two metalloproteases playing a pivotal role in melanoma progression, as direct targets of miR-126&126*. In addition, as ADAM9 and MMP7 share a role in the proteolytic cleavage of the HB-EGF precursor, we looked for the effectiveness of this regulatory pathway in melanoma, confirming the decrease of HB-EGF activation as a consequence of miR-126&126*-dependent downmodulation of ADAM9 and MMP7. Finally, gene profile analyses showed that miR-126&126* reexpression was sufficient to inactivate other key signaling pathways involved in the oncogenic transformation, as PI3K/AKT and MAPK, and to restore melanogenesis, as indicated by KIT/MITF/TYR induction. In view of this miR-126&126* wide-ranging action, we believe that the replacement of these microRNAs might be considered a promising therapeutic approach.

Constitutive activation of the ETS‐1‐miR‐222 circuitry in metastatic melanoma

MicroRNAs‐221 and ‐222 are highly upregulated in several solid tumors, including melanomas. We demonstrate that the proto‐oncogene ETS‐1, involved in the pathogenesis of cancers of different origin, is a transcriptional regulator of miR‐222 by direct binding to its promoter region. Differently from 293FT cells or early stage melanomas, where unphosphorylated ETS‐1 represses miR‐222 transcription, in metastatic melanoma the constitutively Thr‐38 phosphorylated fraction of ETS‐1 induces miR‐222. Despite its stepwise decreased expression along with melanoma progression, the oncogenic activity of ETS‐1 relies on its RAS/RAF/ERK‐dependent phosphorylation status more than on its total amount. To close the loop, we demonstrate ETS‐1 as a direct target of miR‐222, but not miR‐221, showing the novel option of their uncoupled functions. In addition, a spatial redistribution of ETS‐1 protein from the nucleus to the cytoplasm is also evidenced in advanced melanoma cells. Finally, in vivo studies confirmed the contribution of miR‐222 to the increased invasive potential obtained by ETS‐ silencing.

The abrogation of the HOXB7/PBX2 complex induces apoptosis in melanoma through the miR-221&222-c-FOS pathway

Cutaneous melanoma is the fastest increasing cancer worldwide. Although several molecular abnormalities have been associated with melanoma progression, the underlying mechanisms are still largely unknown and few targeted therapies are under evaluation. Here we show that the HOXB7/PBX2 dimer acts as a positive transcriptional regulator of the oncogenic microRNA‐221 and ‐222. In addition, demonstrating c‐FOS as a direct target of miR‐221&222, we identify a HOXB7/PBX2→miR‐221&222 →c‐FOS regulatory link, whereby the abrogation of functional HOXB7/PBX2 dimers leads to reduced miR‐221&222 transcription and elevated c‐FOS expression with consequent cell death. Taking advantage of the treatment with the peptide HXR9, an antagonist of HOX/PBX dimerization, we recognize miR‐221&222 as effectors of its action, in turn confirming the HXR9 efficacy in the treatment of human melanoma malignancy, whilst sparing normal human melanocytes. Our findings, besides suggesting the potential therapeutic of HXR9 or its derivatives in malignant melanoma, suggest the disruption of the HOXB7/PBX2 complexes, miR‐221&222 inhibition or even better their combination, as innovative therapeutic approaches.

The EL2 Rat Fibroblasts Line: Differential Effects of Growth Factors (EGF, PDGF, FGF, TPA and TGFβ) on Cell Proliferation and c‐fos Expressiona

Fibroblasts growth in culture is controlled by the concentration of serum in the medium. Serum contains a growth factor released by platelets during clot formation’*2 and growth factors derived from platelet-poor p l a ~ m a . ~ The platelet-derived growth factor (PDGF) allows cells to become competent to enter the cell cycle, but is not able to drive them into the S phase. Progression through GO/Gl into S after PDGF stimulus is induced by either plasma supplementation or sequential addition of its active constituents, i.e., epidermal growth factor (EGF) and somatomedin C (IGF I).‘ On the basis of these observations, it has been proposed that PDGF and EGF play a different role in the prereplicative phase, acting as a competence and a progression factor respectively. The competence/progression model has been postulated on the basis of studies on BALB/c 3T3 fibroblasts, but it is not established whether it may be generally applied to normal fibroblasts in culture.’ Recent studies on fibroblasts and lymphoid cells indicated that growth factors induce the activation of proto-oncogenes encoding nuclear proteins of unknown function (c-myc, c-myb, c-fos).It has been suggested that these genes may be directly involved in the regulation of cell proliferation, as transducers of growth factor

HOXB1 restored expression promotes apoptosis and differentiation in the HL60 leukemic cell line

BackgroundHomeobox (HOX) genes deregulation has been largely implicated in the development of human leukemia. Among the HOXB cluster, HOXB1 was silent in a number of analyzed acute myeloid leukemia (AML) primary cells and cell lines, whereas it was expressed in normal terminally differentiated peripheral blood cells.MethodsWe evaluated the biological effects and the transcriptome changes determined by the retroviral transduction of HOXB1 in the human promyelocytic cell line HL60.ResultsOur results suggest that the enforced expression of HOXB1 reduces cell growth proliferation, inducing apoptosis and cell differentiation along the monocytic and granulocytic lineages. Accordingly, gene expression analysis showed the HOXB1-dependent down-regulation of some tumor promoting genes, paralleled by the up-regulation of apoptosis- and differentiation-related genes, thus supporting a tumor suppressor role for HOXB1 in AML. Finally, we indicated HOXB1 promoter hypermethylation as a mechanism responsible for HOXB1 silencing.ConclusionsWe propose HOXB1 as an additional member of the HOX family with tumour suppressor properties suggesting a HOXB1/ATRA combination as a possible future therapeutic strategy in AML.

SCD5-induced oleic acid production reduces melanoma malignancy by intracellular retention of SPARC and cathepsin B.

A proper balance between saturated and unsaturated fatty acids (FAs) is required for maintaining cell homeostasis. The increased demand of FAs to assemble the plasma membranes of continuously dividing cancer cells might unbalance this ratio and critically affect tumour outgrowth. We unveiled the role of the stearoyl‐CoA desaturase SCD5 in converting saturated FAs into mono‐unsaturated FAs during melanoma progression. SCD5 is down‐regulated in advanced melanoma and its restored expression significantly reduced melanoma malignancy, both in vitro and in vivo, through a mechanism governing the secretion of extracellular matrix proteins, such as secreted protein acidic and rich in cysteine (SPARC) and collagen IV and of their proteases, such as cathepsin B. Enforced expression of SCD5 or supplementation of its enzymatic product, oleic acid, reduced the intracellular pH (pHe > pHi) and, in turn, vesicular trafficking across plasma membranes as well as melanoma dissemination. This intracellular acidification appears also to depend on SCD5‐induced reduction of the C2 subunit of the vacuolar H+‐ATPase, a proton pump whose inhibition changes the secretion profile of cancer cells. Our data support a role for SCD5 and its enzymatic product, oleic acid, in protection against malignancy, offering an explanation for the beneficial Mediterranean diet. Furthermore, SCD5 appears to functionally connect tumour cells and the surrounding stroma toward modification of the tumour microenvironment, with consequences on tumour spread and resistance to treatment. Copyright

Differential regulation of iron-responsive element-binding protein in activated lymphocytes versus monocytes-macrophages

Note: Department of Hematology-Oncology, Istituto Superiore di Sanita, Rome, Italy. (Invited paper) Reference GR-KUHN-ARTICLE-1991-004 Record created on 2008-02-25, modified on 2017-05-12

Expression of transferrin receptors: differential regulatory mechanisms in monocytes-macrophages versus other hemopoietic cells.

Interaction of a cell membrane receptor with its ligand induces either activation of a specific biological process or cellular uptake of an essential nutrient. On this basis, receptors have been classified in two categories:’ class I receptors transmit a specific piece of information (e.g., epidermal and platelet-derived growth factor receptors modulate the growth of a variety of cell types); class I1 receptors interact with and internalize glycoproteins carrying essential metabolic factors (e.g., low-densitylipoproteins (LDL) and transferrin (Trf) receptors allow respectively the uptake of cholesterol and iron). Upon exposure to ligand, class I receptors are rapidly downregulated. In contrast, binding of ligand with class I1 receptors does not usually lead to modulation of their number. A paradigmatic class I1 receptor is the human receptor for LDL.’ Incubation of cells with cholesterol-saturated LDL results in a decrease in the number of receptors. Conversely, incubation in the absence of LDL causes a rise in their number. Both phenomena are mediated by modulation of receptor synthesis, which is in turn controlled by the intracellular concentration of free cholesterol.2 Recent studies suggest that the Trf receptor may similarly represent a typical class I1 receptor: the level of intracellular iron apparently modulates the rate of Trf receptor synthesis,’ thus resembling the regulation of LDL receptors via cholesterol. All types of cells require iron to sustain essential metabolic pathways. Additionally, actively dividing cells need iron for their growth, presumably because the metal is

AP2α controls the dynamic balance between miR-126&126* and miR-221&222 during melanoma progression

Accumulating evidences have shown the association between aberrantly expressed microRNAs (miRs) and cancer, where these small regulatory RNAs appear to dictate the cell fate by regulating all the main biological processes. We demonstrated the responsibility of the circuitry connecting the oncomiR-221&222 with the tumor suppressors miR-126&126* in melanoma development and progression. According to the inverse correlation between endogenous miR-221&222 and miR-126&126*, respectively increasing or decreasing with malignancy, their enforced expression or silencing was sufficient for a reciprocal regulation. In line with the opposite roles of these miRs, protein analyses confirmed the reverse expression pattern of miR-126&126*-targeted genes that were induced by miR-221&222. Looking for a central player in this complex network, we revealed the dual regulation of AP2α, on one side directly targeted by miR-221&222 and on the other a transcriptional activator of miR-126&126*. We showed the chance of restoring miR-126&126* expression in metastatic melanoma to reduce the amount of mature intracellular heparin-binding EGF like growth factor, thus preventing promyelocytic leukemia zinc finger delocalization and maintaining its repression on miR-221&222 promoter. Thus, the low-residual quantity of these two miRs assures the release of AP2α expression, which in turn binds to and induces miR-126&126* transcription. All together these results point to an unbalanced ratio functional to melanoma malignancy between these two couples of miRs. During progression this balance gradually moves from miR-126&126* toward miR-221&222. This circuitry, besides confirming the central role of AP2α in orchestrating melanoma development and/or progression, further displays the significance of these miRs in cancer and the option of utilizing them for novel therapeutics.

Expression of c‐fos in Human Normal and Neoplastic Monocyte‐Macrophage Differentiation

Recent studies have shed considerable light on the physiological function of proto-oncogenes (c-onc), as well as on the role of viral oncogenes (v-onc) and “deregulated” c-onc in neoplastic transformation.’ Proto-oncogenes encoding nuclear proteins (i.e.. c-myc, c-myb, c-fos) seemingly function as transducers of growth factor signals within the genome, via activation of genes whose products are required for cell proliferation. The human fos gene (i.e.. the cellular homologue of the transforming gene of the FBJ murine osteosarcoma virus’) is localized on chromosome 143 and codes for a 380-aa nuclear protein of yet unknown f ~ n c t i o n . ~ c-fos expression is rapidly activated in quiescent fibroblasts stimulated by serum or growth factors, thus suggesting a role for c-fos in cell-cycle controLsS6 However, other experiments suggest a correlation between c-fos expression and cell differentiation. Thus, fos RNA and/or protein(s) have been detected in both murine and human late gestation extraembryonic tissues:* in murine hemopoietic cells or leukemic lines treated by colony-stimulating factors,’ as well as in human promyelocytic or monoblastic lines induced by phorbol esters.”.”