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Multiple myeloma (MM) is tightly dependent on inflammatory bone marrow microenvironment. IL-17 producing CD4+ T cells (Th17) sustain MM cells growth and osteoclasts-dependent bone damage. In turn, Th17 differentiation relies on inflammatory stimuli. Here, we investigated the role of miR-21 in Th17-mediated MM tumor growth and bone disease. We found that early inhibition of miR-21 in naive T cells (miR-21i-T cells) impaired Th17 differentiation in vitro and abrogated Th17-mediated MM cell proliferation and osteoclasts activity. We validated these findings in NOD/SCID-g-NULL mice, intratibially injected with miR-21i-T cells and MM cells. A Pairwise RNAseq and proteome/phosphoproteome analysis in Th17 cells demonstrated that miR-21 inhibition led to upregulation of STAT-1/-5a-5b, STAT-3 impairment and redirection of Th17 to Th1/Th2 like activated/polarized cells. Our findings disclose the role of miR-21 in pathogenic Th17 activity and open the avenue to the design of miR-21-targeting strategies to counteract microenvironment dependence of MM growth and bone disease.

Multiple myeloma (MM) is a hematologic malignancy characterized by high genomic instability. Here we provide evidence that hyper-activation of DNA ligase III (LIG3) is crucial for genomic instability and survival of MM cells. LIG3 mRNA expression in MM patients correlates with shorter survival and even increases with more advanced stage of disease. Knockdown of LIG3 impairs MM cells viability in vitro and in vivo, suggesting that neoplastic plasmacells are dependent on LIG3-driven repair. To investigate the mechanisms involved in LIG3 expression, we investigated the post-transcriptional regulation. We identified miR-22-3p as effective negative regulator of LIG3 in MM. Enforced expression of miR-22 in MM cells downregulated LIG3 protein, which in turn increased DNA damage inhibiting in vitro and in vivo cell growth. Taken together, our findings demonstrate that myeloma cells are addicted to LIG3, which can be effectively inhibited by miR-22, promoting a novel axis of genome stability regulation.

Dendritic cells (DCs) have a key role in regulating tumor immunity, tumor cell growth and drug resistance. We hypothesized that multiple myeloma (MM) cells might recruit and reprogram DCs to a tumor-permissive phenotype by changes within their microRNA (miRNA) network. By analyzing six different miRNA-profiling data sets, miR-29b was identified as the only miRNA upregulated in normal mature DCs and significantly downregulated in tumor-associated DCs. This finding was validated in primary DCs co-cultured in vitro with MM cell lines and in primary bone marrow DCs from MM patients. In DCs co-cultured with MM cells, enforced expression of miR-29b counteracted pro-inflammatory pathways, including signal transducer and activator of transcription 3 and nuclear factor-κ B, and cytokine/chemokine signaling networks, which correlated with patients' adverse prognosis and development of bone disease. Moreover, miR-29b downregulated interleukin-23 in vitro and in the SCID-synth-hu in vivo model, and antagonized a Th17 inflammatory response. All together, these effects translated into strong anti-proliferative activity and reduction of genomic instability of MM cells. Our study demonstrates that MM reprograms the DCs functional phenotype by downregulating miR-29b whose reconstitution impairs DCs ability to sustain MM cell growth and survival. These results underscore miR-29b as an innovative and attractive candidate for miRNA-based immune therapy of MM.

// Maria Rita Pitari 1 , Marco Rossi 1 , Nicola Amodio 1 , Cirino Botta 1 , Eugenio Morelli 1 , Cinzia Federico 1 , Annamaria Gulla 1 , Daniele Caracciolo 1 , Maria Teresa Di Martino 1 , Mariamena Arbitrio 2 , Antonio Giordano 3, 4 , Pierosandro Tagliaferri 1 , Pierfrancesco Tassone 1, 4 1 Department of Experimental and Clinical Medicine and T. Campanella Cancer Center, Magna Graecia University, S. Venuta University Campus, Catanzaro, Italy 2 ISN-CNR, Roccelletta di Borgia, Catanzaro, Italy 3 Department of Human Pathology and Oncology, University of Siena, Siena, Italy 4 Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, College of Science and Technology, Temple University, Philadelphia, PA, USA Correspondence to: Pierfrancesco Tassone, e-mail: tassone@unicz.it Keywords: miR-21, miRNAs, multiple myeloma bone disease, OPG, RANKL Received: May 05, 2015 Accepted: June 11, 2015 Published: June 24, 2015 ABSTRACT miR-21 is an oncogenic microRNA (miRNA) with an emerging role as therapeutic target in human malignancies, including multiple myeloma (MM). Here we investigated whether miR-21 is involved in MM-related bone disease (BD). We found that miR-21 expression is dramatically enhanced, while osteoprotegerin (OPG) is strongly reduced, in bone marrow stromal cells (BMSCs) adherent to MM cells. On this basis, we validated the 3′UTR of OPG mRNA as miR-21 target. Constitutive miR-21 inhibition in lentiviral-transduced BMSCs adherent to MM cells restored OPG expression and secretion. Interestingly, miR-21 inhibition reduced RANKL production by BMSCs. Overexpression of protein inhibitor of activated STAT3 (PIAS3), which is a direct and validated target of miR-21, antagonized STAT3-mediated RANKL gene activation. Finally, we demonstrate that constitutive expression of miR-21 inhibitors in BMSCs restores RANKL/OPG balance and dramatically impairs the resorbing activity of mature osteoclasts. Taken together, our data provide proof-of-concept that miR-21 overexpression within MM-microenviroment plays a crucial role in bone resorption/apposition balance, supporting the design of innovative miR-21 inhibition-based strategies for MM-related BD.

// Lavinia Raimondi 1 , Nicola Amodio 1 , Maria Teresa Di Martino 1 , Emanuela Altomare 1 , Marzia Leotta 1 , Daniele Caracciolo 1 , Annamaria Gulla 1 , Antonino Neri 2 , Simona Taverna 3 , Patrizia D’Aquila 4 , Riccardo Alessandro 3 , Antonio Giordano 5 , Pierosandro Tagliaferri 1 and Pierfrancesco Tassone 1,5 . 1 Department of Experimental and Clinical Medicine, Magna Graecia University and Medical Oncology Unit, T. Campanella Cancer Center, Salvatore Venuta University Campus, Catanzaro, Italy 2 Department of Medical Sciences University of Milan, Hematology1, IRCCS Policlinico Foundation, Milan, Italy 3 Department of Pathology and Forensic and Medical Biotechnology, Section of Biology and Genetics, University of Palermo, Italy 4 Department of Biology, Ecology and Earth Science (DiBEST),University of Calabria, Arcavacata di Rende, Cosenza, Italy 5 Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, College of Science and Technology, Temple University, Philadelphia, PA, USA Correspondence: Pierfrancesco Tassone, email: // Keywords : miR-199-5p, microRNA, miRNA, multiple myeloma, plasma cell leukemia, microenviroment, hypoxia, angiogenesis Received : December 27, 2013 Accepted : March 12, 2014 Published : March 14, 2014 Abstract Multiple myeloma (MM) cells induce relevant angiogenic effects within the human bone marrow milieu (huBMM) by the aberrant expression of angiogenic factors. Hypoxia triggers angiogenic events within the huBMM and the transcription factor hypoxia-inducible factor-1α (HIF-1α) is over-expressed by MM cells. Since synthetic miR-199a-5p mimics negatively regulates HIF-1α, we here investigated a miRNA-based therapeutic strategy against hypoxic MM cells. We indeed found that enforced expression of miR-199a-5p led to down-modulated expression of HIF-1α as well as of other pro-angiogenic factors such as VEGF-A, IL-8, and FGFb in hypoxic MM cells in vitro . Moreover, miR-199a-5p negatively affected MM cells migration, while it increased the adhesion of MM cells to bone marrow stromal cells (BMSCs) in hypoxic conditions. Furthermore, transfection of MM cells with miR-199a-5p significantly impaired also endothelial cells migration and down-regulated the expression of endothelial adhesion molecules such as VCAM-1 and ICAM-1. Finally, we identified a hypoxia\AKT/miR-199a-5p loop as a potential molecular mechanism responsible of miR-199a-5p down-regulation in hypoxic MM cells. Taken together our results indicate that miR-199a-5p has an important role for the pathogenesis of MM and support the hypothesis that targeting angiogenesis via a miRNA/HIF-1α pathway may represent a novel potential therapeutical approach for this still lethal disease.

Multiple myeloma (MM) is an incurable plasma cell neoplasm accounting for about 10% of all hematologic malignancies. Recently, emerging evidence is disclosing the complexity of bone marrow interactions between MM cells and infiltrating immune cells, which have been reported to promote proliferation, survival and drug resistance of tumor cells. MicroRNAs (miRNAs) are small non-coding RNA molecules with regulatory functions in the cell, whose expression has predictive and prognostic value in different malignancies. MiRNAs are gaining increasing interest due to their capability to polarize the immune-response through different mechanisms, which include the molecular reprogramming of immune cells. This characteristic, together with the antitumor activity of miRNA mimics or inhibitors, make the miRNA network an attractive area of investigation for novel anti-MM therapeutic approaches. In this review, we will discuss the recent advances in the understanding of the interplay between MM cells and bone marrow resident immune cells, with special focus on the molecular and functional changes induced by miRNA network modulation. We will finally indicate potential targets for therapeutic intervention.