Lisabianca Bottero

Caveolin-1 tumor-promoting role in human melanoma

Caveolin‐1 (Cav‐1), a member of the caveolin family, regulates caveolae‐associated signaling proteins, which are involved in many biological processes, including cancer development. Cav‐1 was found to exert a complex and ambiguous role as oncogene or tumor suppressor depending on the cellular microenvironment. Here we investigated Cav‐1 expression and function in a panel of melanomas, finding its expression in all the cell lines. The exception was the primary vertical melanoma cell line, WM983A, characterized by the lack of Cav‐1, and then utilized as a recipient for Cav‐1 gene transduction to address a series of functional studies. The alleged yet controversial role of phospho (Ph)‐Cav‐1 on cell regulation was also tested by transducing the nonphosphorylatable Cav‐1Y14A mutant. Wild‐type Cav‐1, but not mutated Cav‐1Y14A, increased tumorigenicity as indicated by enhanced proliferation, migration, invasion and capacity of forming foci in semisolid medium. Accordingly, Cav‐1 silencing inhibited melanoma cell growth reducing some of the typical traits of malignancy. Finally, we detected a secreted fraction of Cav‐1 associated with cell released microvesicular particles able to stimulate in vitro anchorage independence, migration and invasion in a paracrine/autocrine fashion and, more important, competent to convey metastatic asset from the donor melanoma to the less aggressive recipient cell line. A direct correlation between Cav‐1 levels, the amount of microvesicles released in the culture medium and MMP‐9 expression was also observed.

Activating mutations in RRAS underlie a phenotype within the RASopathy spectrum and contribute to leukaemogenesis

RASopathies, a family of disorders characterized by cardiac defects, defective growth, facial dysmorphism, variable cognitive deficits and predisposition to certain malignancies, are caused by constitutional dysregulation of RAS signalling predominantly through the RAF/MEK/ERK (MAPK) cascade. We report on two germline mutations (p.Gly39dup and p.Val55Met) in RRAS, a gene encoding a small monomeric GTPase controlling cell adhesion, spreading and migration, underlying a rare (2 subjects among 504 individuals analysed) and variable phenotype with features partially overlapping Noonan syndrome, the most common RASopathy. We also identified somatic RRAS mutations (p.Gly39dup and p.Gln87Leu) in 2 of 110 cases of non-syndromic juvenile myelomonocytic leukaemia, a childhood myeloproliferative/myelodysplastic disease caused by upregulated RAS signalling, defining an atypical form of this haematological disorder rapidly progressing to acute myeloid leukaemia. Two of the three identified mutations affected known oncogenic hotspots of RAS genes and conferred variably enhanced RRAS function and stimulus-dependent MAPK activation. Expression of an RRAS mutant homolog in Caenorhabditis elegans enhanced RAS signalling and engendered protruding vulva, a phenotype previously linked to the RASopathy-causing SHOC2S2G mutant. Overall, these findings provide evidence of a functional link between RRAS and MAPK signalling and reveal an unpredicted role of enhanced RRAS function in human disease.

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.

Role of PLZF in melanoma progression

The promyelocytic leukemia zinc finger (PLZF) protein has been described as a transcriptional repressor of homeobox (HOX)-containing genes during embryogenesis. As we previously demonstrated a functional link between overexpression of HOXB7 and melanoma progression, we investigated the lack of PLZF as the possible cause of HOXB7 constitutive activation in these neoplastic cells. Accordingly, we found PLZF expression in melanocytes, but not in melanoma cells, a pattern inversely related to that of HOXB7. PLZF retroviral gene transduction was then performed in a panel of melanoma cell lines, and tumorigenicity was compared with that of empty vector-transduced control cell lines. Evaluation of in vitro migration, invasion and adhesion indicated that PLZF gene transduction induced a less malignant phenotype, as confirmed through in vivo studies performed in athymic nude mice. This reduced tumorigenicity was not coupled with HOXB7 repression. In order to find more about the molecular targets of PLZF, the gene expression profiles of PLZF- and empty vector-transduced A375 melanoma cells were analysed by Atlas Cancer macroarray. Among several genes modulated by PLZF enforced expression, of particular interest were integrin αvβ3, osteonectin/SPARC and matrix metalloprotease-9 that were downmodulated, and the tyrosinase-related protein-1 that was upregulated in all the analysed samples. This profile confirms the reduced tumorigenic phenotype with reversion to a more differentiated, melanocyte like, pattern, thus suggesting a suppressor role for PLZF in solid tumors. Moreover, these results indicate that PLZF and HOXB7 are functionally independent and that their coupled deregulation may account for most of the alterations described in melanomas.

Expression of protein kinase C genes during ontogenic development of the central nervous system.

We have analyzed the RNA expression of three protein kinase C (PKC) genes (alpha, beta, and gamma) in human and murine central nervous systems during embryonic-fetal, perinatal, and adult life. Analysis of human brain poly(A)+ RNA indicates that expression of PKC alpha and beta genes can be detected as early as 6 weeks postconception, undergoes a gradual increase until 9 weeks postconception, and reaches its highest level in the adult stage, and that the PKC gamma gene, although not expressed during embryonic and early fetal development, is abundantly expressed in the adult period. Similar developmental patterns were observed in human spinal cord and medulla oblongata. A detailed analysis of PKC gene expression during mammalian ontogeny was performed on poly(A)+ RNA from the brain cells of murine embryos at different stages of development and the brain cells of neonatal and adult mice. The ontogenetic patterns were similar to those observed for human brain. Furthermore, we observed that the expression of PKC gamma is induced in the peri- and postnatal phases. These results suggest that expression of PKC alpha, beta, and gamma genes possibly mediates the development of central neuronal functions, and expression of PKC gamma in particular may be involved in the development of peri- and postnatal functions.

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.

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

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