Gianfranco Mattia

HOXB7 constitutively activates basic fibroblast growth factor in melanomas.

Homeobox (HOX) genes control axial specification during mammalian development and also regulate skin morphogenesis. Although selected HOX genes are variably expressed in leukemias and kidney and colon cancer cell lines, their relationship with the neoplastic phenotype remains unclear. In both normal development and neoplastic transformation, HOX target genes are largely unknown. We investigated the expression and function of HOXB cluster genes in human melanoma. The HOXB7 gene was constitutively expressed in all 25 melanoma cell lines and analyzed under both normal and serum-starved conditions, as well as in in vivo primary and metastatic melanoma cells; conversely, HOXB7 was expressed in proliferating but not quiescent normal melanocytes. Treatment of melanoma cell lines with antisense oligomers targeting HOXB7 mRNA markedly inhibited cell proliferation and specifically abolished expression of basic fibroblast growth factor (bFGF) mRNA. Band shift and cotransfection experiments showed that HOXB7 directly transactivates the hFGF gene through one out of five putative homeodomain binding sites present in its promoter. These novel findings indicate a key role for constitutive HOXB7 expression in melanoma cell proliferation via bFGF. The results also raise the possibility that growth factor genes are critical HOX target genes in other developmental and/or neoplastic cell systems.

miR‐34a predicts survival of Ewing's sarcoma patients and directly influences cell chemo‐sensitivity and malignancy

Identification of factors to detect chemotherapy‐resistant tumours at diagnosis is a first priority for risk‐adapted therapy in the oncology of children and young adults, where more individualized, effective, and less toxic treatments are highly desirable. In this study, we analysed the miRNAs discriminating Ewings sarcoma (EWS) patients with different clinical outcomes in order to identify new indicators of prognosis. miRNA expression was investigated in 49 primary EWSs by using the Agilent human miRNA microarray v.2 and/or qRT‐PCR. Statistical power of the samples studied for miRNA expression was verified, indicating adequate sample size. Microarray analysis defined a signature of five miRNAs (miR‐34a, miR‐23a, miR‐92a, miR‐490‐3p, and miR‐130b) as an independent predictor of risk for disease progression and survival. Validation analysis in the extended sample set indicated that both miR‐34a and miR‐490‐3p achieved sufficient statistical power to predict prognosis. Results were particularly robust for miR‐34a, which appeared associated with either event‐free or overall survival and emerged as a significant predictor also after multivariate analysis. Patients with the highest expression of miR‐34a did not experience adverse events in 5 years; in contrast, patients with the lowest expression recurred within 2 years. High expression of miR34a can be detected also in paraffin‐embedded tissues by in situ hybridization, thus contributing to an easy routine evaluation of this miRNA. Functional analysis of miR‐34a in EWS cell lines indicated that when miR‐34a expression was enforced, cells were less proliferative, less malignant, and sensitized to doxorubicin and vincristine. Expression of miR‐34a could be increased in p53wt cells by treatment with nutlin‐3a. Accordingly, nutlin‐3a synergizes with doxorubicin. Overall, our data indicate that miR‐34a expression is a strong predictor of outcome in EWS. Restoration of miR‐34a activity may be useful to decrease malignancy and increase tumour sensitivity to current drugs, so sparing excessive long‐term toxicity to EWS patients. Copyright

Transduction of the SkBr3 breast carcinoma cell line with the HOXB7 gene induces bFGF expression, increases cell proliferation and reduces growth factor dependence

Several melanomas, carcinomas, glioblastomas and leukemias showed coordinated expression of HOXB7 and bFGF with exception of the SkBr3 mammary carcinoma that was negative for both. Transduction of HOXB7 gene into SkBr3 cells, induced bFGF expression, increased growth rate, independence from serum withdrawal and ability to form colonies in semisolid medium. ELISA assay showed that most of bFGF was associated to cell lysate when cells were cultured at 1% serum whereas in cells kept to 10% serum bFGF was detected both within cell lysate or secreted into cell supernatants. Antisense oligos to bFGF inhibited the growth of cells cultured in 1%, indicating that beside the possible activation of additional genes other than bFGF by HOXB7 transduction, only bFGF induction accounts for the observed results. Moreover, since inhibition of cell proliferation occurred in cells kept in 1% but not 10% serum, a bFGF intracrine loop appears operative in serum starved SkBr3/HOXB7 cells. Also, these results further indicate bFGF as target of HOXB7.

MicroRNA-221 and -222 pathway controls melanoma progression.

MicroRNAs (miRNAs) represent a new family of small noncoding RNAs that negatively regulate gene expression. Recent studies demonstrated miRNA involvement in all the main biological processes, including tumor development as a consequence of an aberrant deregulated expression. Growing evidence is showing the capability of miRNA expression profiles to unequivocally distinguish between normal and neoplastic tissues, leading to the identification of new diagnostic and/or prognostic molecular markers. In addition, miRNAs might eventually represent new targets to aim at as innovative therapeutic approaches, particularly relevant in those types of cancer, such as melanoma, which are still lacking effective traditional therapies. In particular, the inhibition of miRNA-221 and -222, which are abnormally expressed in melanoma and favor the induction of the malignant phenotype by downregulating c-KIT receptor and p27Kip, might in the future represent an efficient treatment for translation into the clinical setting.

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.

Enforced expression of HOXB7 promotes hematopoietic stem cell proliferation and myeloid-restricted progenitor differentiation.

Hematopoietic progenitor/stem cells (HPCs/HSCs) purified from human adult peripheral blood (PB) were triggered into cycling, retrovirally transduced with HOXB7 and then functionally assayed in vitro. HPCs were assayed in multi- and unilineage differentiation cultures in either liquid phase or semisolid medium, primitive HPCs in the high proliferative potential colony-forming cell (HPP – CFC) evaluation system and putative HSCs in Dexter type long-term culture (LTC) as LTC initiating cells (LTC – ICs). Control experiments ensured that the exogenous HOXB7 gene was constantly expressed, while the endogenous one was barely or not transcribed. Enforced expression of the gene markedly modulated the proliferation/differentiation program of the entire HSC/HPC population. Enforced HOXB7 expression exerted a potent stimulatory effect on the proliferation of the primitive HPC and putative HSC subsets, assayed as HPP – CFCs and LTC – ICs respectively. While not modifying the total number of HPCs, exogenous HOXB7 induced an increase of the number of granulo-monocytic (GM) HPCs [colony-forming unit GM (CFU – GM) CFU – GM, CFU-G and CFU-M, as evaluated by clonogenic assays] and markedly amplified the progeny of both CFU-G and CFU-M, which showed a sustained proliferation through at least 1 – 2 months (as evaluated in liquid suspension culture). The prolonged proliferative stimulus induced by HOXB7 transfer into LTC, primitive and GM oriented HPC culture was characterized by persistent proliferation of a discrete population of blast cells and a large pool of differentiated myeloid precursors. Altogether, these results suggest the hypothesis that the proliferative stimulus exerted by exogenous HOXB7 in primitive and GM-oriented HPCs may represent a preleukemic immortalization step. Consistent with the functional role of HOXB7 in the initial ontogenetic phase, these studies indicate that ectopic HOXB7 expression in early HPCs and HSCs from adult PB stimulates their self renewal, sustained proliferation and myeloid differentiation.

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.

Factor-V expression in platelets from human megakaryocytic culture.

The origin of platelet‐factor‐V has long been discussed. To elucidate whether and when human platelet‐factor‐V is synthesized by megakaryocytes, we utilized in vitro‐generated megakaryocytes capable of producing platelets. Factor‐V gene was silent in purified progenitors and megakaryocytic precursors but was expressed in late culture phase and maintained also in platelets. Similarly, factor‐V protein was expressed in mature proplatelet‐bearing megakaryocytes (immunofluorescence analysis); it was also detectable in cultured megakaryocytes and platelets (Western blotting) and within permeabilized cultured platelets (flow cytometry). The absence of other cells in our culture system indicates conclusively that human megakaryocytes synthesize factor‐V.