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Introduction Recently, the Food and Drug Administration authorized the marketing of IQOS Tobacco Heating System as a Modified Risk Tobacco Product based on an electronic heat-not-burn technology that purports to reduce the risk. Methods Sprague-Dawley rats were exposed in a whole-body mode to IQOS aerosol for 4 weeks. We performed the chemical characterization of IQOS mainstream and we studied the ultrastructural changes in trachea and lung parenchyma of rats exposed to IQOS stick mainstream and tissue pro-inflammatory markers. We investigated the reactive oxygen species amount along with the markers of tissue and DNA oxidative damage. Moreover, we tested the putative genotoxicity of IQOS mainstream through Ames and alkaline Comet mutagenicity assays. Results Here, we identified irritating and carcinogenic compounds including aldehydes and polycyclic aromatic hydrocarbons in the IQOS mainstream as sign of incomplete combustion and degradation of tobacco, that lead to severe remodelling of smaller and largest rat airways. We demonstrated that IQOS mainstream induces lung enzymes that activate carcinogens, increases tissue reactive radical concentration; promotes oxidative DNA breaks and gene level DNA damage; and stimulates mitogen activated protein kinase pathway which is involved in the conventional tobacco smoke-induced cancer progression. Conclusions Collectively, our findings reveal that IQOS causes grave lung damage and promotes factors that increase cancer risk. Implications IQOS has been proposed as a safer alternative to conventional cigarettes, due to depressed concentration of various harmful constituents typical of traditional tobacco smoke. However, its lower health risks to consumers have yet to be determined. Our findings confirm that IQOS mainstream contains pyrolysis and thermogenic degradation by-products, the same harmful constituents of traditional cigarette smoke, and, for the first time, we show that it causes grave lung damage and promotes factors that increase cancer risk in the animal model.

Autophagy is a catabolic pathway activated in response to different cellular stressors, such as damaged organelles, accumulation of misfolded or unfolded proteins, ER stress, accumulation of reactive oxygen species, and DNA damage. Some DNA damage sensors like FOXO3a, ATM, ATR, and p53 are known to be important autophagy regulators, and autophagy seems therefore to have a role in DNA damage response (DDR). Recent studies have partly clarified the pathways that induce autophagy during DDR, but its precise role is still not well known. Previous studies have shown that autophagy alterations induce an increase in DNA damage and in the occurrence of tumor and neurodegenerative diseases, highlighting its fundamental role in the maintenance of genomic stability. During DDR, autophagy could act as a source of energy to maintain cell cycle arrest and to sustain DNA repair activities. In addition, autophagy seems to play a role in the degradation of components involved in the repair machinery. In this paper, molecules which are able to induce oxidative stress and/or DNA damage have been selected and their toxic and genotoxic effects on the U937 cell line have been assessed in the presence of the single compounds and in concurrence with an inhibitor (chloroquine) or an inducer (rapamycin) of autophagy. Our data seem to corroborate the fundamental role of this pathway in response to direct and indirect DNA-damaging agents. The inhibition of autophagy through chloroquine had no effect on the genotoxicity induced by the tested compounds, but it led to a high increase of cytotoxicity. The induction of autophagy, through cotreatment with rapamycin, reduced the genotoxic activity of the compounds. The present study confirms the cytoprotective role of autophagy during DDR; its inhibition can sensitize cancer cells to DNA-damaging agents. The modulation of this pathway could therefore be an innovative approach able to reduce the toxicity of many compounds and to enhance the activity of others, including anticancer drugs.

Introduction: Electronic cigarettes (e-cigs) are devices thought to deliver nicotine in a vaping solution without tobacco combustion. Perceived as a safer alternative to conventional cigarettes, e-cigs are aggressively marketed as lifestyle-choice consumables, thanks to few regulatory restrictions. E-cigs have also gained popularity among never-smokers and teenagers, becoming an emergent public health issue. Despite the burgeoning worldwide consumption, their safety remains largely unproven and it is unknown whether these devices could cause in vivo toxicological effects contributing to cancer. Materials and methods: Male Sprague Dawley rats were randomly split into two groups: non-exposed and whole-body mode exposed (10 animals per group). Treated animals consumed 1 mL/day of e-liquid containing 18 mg/mL of nicotine. During the experiment the e-cigarette voltage was set at 5.5 (and the wattage was about 15). Animals were exposed for 4 hours/day for 5 consecutive days/week, and for 4 consecutive weeks. Results: Toxic aldehydes production, due to thermal decomposition of glycerol, were detected in e-cig aerosol. In exposed lung, we observed a significant increase in phase I CYP1A1/2, CYP2B1/2, CY2C11 and CYP3A enzymes. The overproduction of reactive oxygen species resulting from CYP induction, together with the free radicals present in the aerosol, was confirmed by means “Electron paramagnetic resonance-radical probe” technique. Notably, we observed that the lung antioxidant enzymes catalase, DT-diaphorase, superoxide dismutase and the conjugating phase II glutathione S-transferases were all markedly reduced in animal treated. In addition, we found a markedly reduced FRAP (ferric reducing antioxidant power) value in the lung of e-cig rodents. Moreover, plasma FRAP levels and measurement of carbonyl residues, as biomarkers of oxidative injury to proteins, were inversely correlated in e-cig vapour-exposed rats. We also found that 8-hydro-2′-deoxyguanosine markedly increased in the lungs of treated rats. Interestingly, e-cigs produce extensive DNA damage in leukocytes (Comet test). We also found that e-cig vapour determines an increase in the percentage of immature micronucleated reticulocytes over normal reticulocyte. Next, the urine of e-cig-exposed animals induced a dose-dependent increase in the number of S. typhimurium revertants in different strains. Discussion and conclusion: We found that e-cigs have a powerful booster effect on phase-I carcinogen-bioactivating enzymes, and increase oxygen free radical production and DNA oxidation to 8-hydroxy- 2′-deoxyguanosine. Furthermore, we found that e-cigs damage DNA not only at chromosomal level in peripheral blood, such as strand breaks in leucocytes and micronuclei formation in reticulocytes, but also at gene level such as point mutations in urine. As these detrimental phenomena are typically induced by conventional cigarettes, the erroneous belief that e-cigs are safe should be retracted and suitable measures implemented to protect public health. Our study should be seen as the starting point for further investigations designed to confirm the harmful health impact of e-cigs, particularly after long-term exposure and under different usage conditions.

In early diabetes, hyperglycemia and the associated metabolic dysregulation promote early changes in the functional properties of cardiomyocytes, progressively leading to the appearance of the diabetic cardiomyopathy phenotype. Recently, the interplay between histone acetyltransferases (HAT) and histone deacetylases (HDAC) has emerged as a crucial factor in the development of cardiac disorders. The present study evaluates whether HDAC inhibition can prevent the development of cardiomyocyte contractile dysfunction induced by a short period of hyperglycemia, with focus on the potential underlying mechanisms. Cell contractility and calcium dynamics were measured in unloaded ventricular myocytes isolated from the heart of control and diabetic rats. Cardiomyocytes were either untreated or exposed to the pan-HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) for 90 min. Then, a fraction of each group of cells was used to evaluate the expression levels of proteins involved in the excitation&ndash, contraction coupling, and the cardiomyocyte metabolic activity, ATP content, and reactive oxygen species levels. SAHA treatment was able to counteract the initial functional derangement in cardiomyocytes by reducing cell oxidative damage. These findings suggest that early HDAC inhibition could be a promising adjuvant approach for preventing diabetes-induced cardiomyocyte oxidative damage, which triggers the pro-inflammatory signal cascade, mitochondrial damage, and ventricular dysfunction.

The chondroitin sulfate proteoglycan 4 ( CSPG4) gene encodes a transmembrane proteoglycan (PG) constituting the largest and most structurally complex macromolecule of the human surfaceome. Its transcript shows an extensive evolutionary conservation and, due to the elaborated intracellular processing of the translated protein, it generates an array of glycoforms with the potential to exert variant-specific functions. CSPG4-mediated molecular events are articulated through the interaction with more than 40 putative ligands and the concurrent involvement of the ectodomain and cytoplasmic tail. Alternating inside-out and outside-in signal transductions may thereby be elicited through a tight functional connection of the PG with the cytoskeleton and its regulators. The potential of CSPG4 to influence both types of signaling mechanisms is also asserted by its lateral mobility along the plasma membrane and its intersection with microdomain-restricted internalization and endocytic trafficking. Owing to the multitude of molecular interplays that CSPG4 may engage, and thanks to a differential phosphorylation of its intracellular domain accounted by crosstalking signaling pathways, the PG stands out for its unique capability to affect numerous cellular phenomena, including those purporting pathologic conditions. We discuss here the progresses made in advancing our understanding about the structural-functional bases for the ability of CSPG4 to widely impact on cell behavior, such as to highlight how its multivalency may be exploited to interfere with disease progression.-Tamburini, E., Dallatomasina, A., Quartararo, J., Cortelazzi, B., Mangieri, D., Lazzaretti, M., Perris, R. Structural deciphering of the NG2/CSPG4 proteoglycan multifunctionality.

Mutations in nuclear genes associated with defective coenzyme A biosynthesis have been identified as responsible for some forms of neurodegeneration with brain iron accumulation (NBIA), namely PKAN and CoPAN. PKAN are defined by mutations in PANK2, encoding the pantothenate kinase 2 enzyme, that account for about 50% of cases of NBIA, whereas mutations in CoA synthase COASY have been recently reported as the second inborn error of CoA synthesis leading to CoPAN. As reported previously, yeast cells expressing the pathogenic mutation exhibited a temperature-sensitive growth defect in the absence of pantothenate and a reduced CoA content. Additional characterization revealed decreased oxygen consumption, reduced activities of mitochondrial respiratory complexes, higher iron content, increased sensitivity to oxidative stress and reduced amount of lipid droplets, thus partially recapitulating the phenotypes found in patients and establishing yeast as a potential model to clarify the pathogenesis underlying PKAN and CoPAN diseases.

Electronic cigarettes (e-cigs) are devices designed to deliver nicotine in a vaping solution rather than smoke and without tobacco combustion. Perceived as a safer alternative to conventional cigarettes, e-cigs are aggressively marketed as lifestyle-choice consumables, thanks to few restrictions and a lack of regulatory guidelines. E-cigs have also gained popularity among never-smokers and teenagers, becoming an emergent public health issue. Despite the burgeoning worldwide consumption of e-cigs, their safety remains largely unproven and it is unknown whether these devices cause in vivo toxicological effects that could contribute to cancer. Here we demonstrate the co-mutagenic and cancer-initiating effects of e-cig vapour in a rat lung model. We found that e-cigs have a powerful booster effect on phase-I carcinogen-bioactivating enzymes, including activators of polycyclic aromatic hydrocarbons (PAHs), and increase oxygen free radical production and DNA oxidation to 8-hydroxy-2′-deoxyguanosine. Furthermore, we found that e-cigs damage DNA not only at chromosomal level in peripheral blood, such as strand breaks in leucocytes and micronuclei formation in reticulocytes, but also at gene level such as point mutations in urine. Our results demonstrate that exposure to e-cigs could endanger human health, particularly among younger more vulnerable consumers.

Low bone marrow oxygen tension and hypoxia-inducible factor-1α overexpression characterize patients with multiple myeloma: role on the transcriptional and proangiogenic profiles of CD138 + cells

Nitroheterocyclic compounds are widely used as therapeutic agents against a variety of protozoan and bacterial infections. However, the literature on these compounds, suspected of being carcinogens, is widely controversial. In this study, cytotoxic and genotoxic potential of three drugs, Nifurtimox (NFX), Benznidazole (BNZ), and Metronidazole (MTZ) was re-evaluated by different assays. Only NFX reduces survival rate in actively proliferating cells. The compounds are more active for base-pair substitution than frameshift induction in Salmonella; NFX and BNZ are more mutagenic than MTZ; they are widely dependent from nitroreduction whereas microsomal fraction S9 weakly affects the mutagenic potential. Comet assay detects BNZ- and NFX-induced DNA damage at doses in the range of therapeutically treated patient plasma concentration; BNZ seems to mainly act through ROS generation whereas a dose-dependent mechanism of DNA damaging is suggested for NFX. The lack of effects on mammalian cells for MTZ is confirmed also in MN assay whereas MN induction is observed for NFX and BNZ. The effects of MTZ, that shows comparatively low reduction potential, seem to be strictly dependent on anaerobic/hypoxic conditions. Both NFX and BNZ may not only lead to cellular damage of the infective agent but also interact with the DNA of mammalian cells.

Osteoblast impairment occurs within multiple myeloma cell infiltration into the bone marrow. Canonical Wnt signaling activation in osteoprogenitor cells is involved in osteoblast formation through the stabilization of dephosphorylated β-catenin and its nuclear translocation. The effects of multiple myeloma cells on Wnt signaling in human mesenchymal/osteoprogenitor cells are unclear. In 60 multiple myeloma patients checked, we found that among the Wnt inhibitors, Dickkopf-1 and secreted frizzled-related protein-3 were produced by multiple myeloma cells. However, although multiple myeloma cells or multiple myeloma bone marrow plasma affected expression of genes in the canonical Wnt signaling and inhibited β-catenin stabilization in murine osteoprogenitor cells, they failed to block the canonical Wnt pathway in human mesenchymal or osteoprogenitor cells. Consistently, Wnt3a stimulation in human osteoprogenitor cells did not blunt the inhibitory effect of multiple myeloma cells on osteoblast formation. Consequently, despite the higher Wnt antagonist bone marrow levels in osteolytic multiple myeloma patients compared with nonosteolytic ones, β-catenin immunostaining was not significantly different. Our results support the link between the production of Wnt antagonists by multiple myeloma cells and the presence of bone lesions in multiple myeloma patients but show that myeloma cells do not inhibit canonical Wnt signaling in human bone microenvironment. [Cancer Res 2007;67(16):7665–74]

Osteopontin (OPN) is a multifunctional bone matrix glycoprotein that is involved in angiogenesis, cell survival and tumor progression. In this study we show that human myeloma cells directly produce OPN and express its major regulating gene Runx2/Cbfa1. The activity of Runx2/Cbfa1 protein in human myeloma cells has also been demonstrated. Moreover, using small interfering RNA (siRNA) to silent Runx2 in myeloma cells, we suppressed OPN mRNA and protein expression. OPN production in myeloma cells was stimulated by growth factors as IL-6 and IFG-1 and in turn OPN stimulated myeloma cell proliferation. In an 'in vitro' angiogenesis system we showed that OPN production by myeloma cells is critical for the proangiogenic effect of myeloma cells. The expression of OPN by purified bone marrow (BM) CD138(+) cells has also been investigated in 60 newly diagnosed multiple myeloma (MM) patients, finding that 40% of MM patients tested expressed OPN. Higher OPN levels have been detected in the BM plasma of MM patients positive for OPN as compared to controls. Moreover, significantly higher BM angiogenesis has been observed in MM patients positive for OPN as compared to those negative. Our data highlight that human myeloma cells with active Runx2/Cbfa1 protein directly produce OPN that is involved in the pathophysiology of MM-induced angiogenesis.

Decreased bone formation contributes to the development of bone lesions in multiple myeloma (MM) patients. In this study, we have investigated the effects of myeloma cells on osteoblast formation and differentiation and the potential role of the critical osteoblast transcription factor RUNX2/CBFA1 (Runt-related transcription factor 2/core-binding factor Runt domain α subunit 1) in the inhibition of osteoblastogenesis in MM. We found that human myeloma cells suppress the formation of human osteoblast progenitors in bone marrow (BM) cultures. Moreover, an inhibitory effect on osteocalcin, alkaline phosphatase, collagen I mRNA, protein expression, and RUNX2/CBFA1 activity by human preosteoblastic cells was observed in cocultures with myeloma cells. The inhibitory effect was more pronounced in the cell-to-cell contact conditions compared with those without the contact and involved the very late antigen 4 (VLA-4) integrin system. Among the soluble osteoblast inhibitors screened, we show the potential contribution of interleukin-7 (IL-7) in the inhibitory effect on osteoblast formation and RUNX2/CBFA1 activity by human myeloma cells in coculture. Finally, our in vitro results were supported in vivo by the finding of a significant reduction in the number of Runx2/Cbfa1-positive cells in the BM biopsies of patients with MM who had osteolytic lesions compared with those who did not have bone lesions, suggesting the critical involvement of RUNX2/CBFA1 in the decreased bone formation in MM. (Blood. 2005;106:2472-2483)

Adenoid cystic carcinoma (ACC) is an indolent tumor that pursues a protracted clinical course with recurrences and late metastases. The aim of this study was to investigate immunohistochemically the expression of p53, bcl-2 protein, and Ki-67 in 21 cases of ACC of the palate, all with a minimum of 10 years and a maximum of 22 years of clinical follow-up. These results were also analyzed with regard to different clinical prognoses of the histologic subtypes of ACC. High expression of p53 and bcl-2 was noted in 19 out of 21 ACC cases (90%), in which most tumor cells (from 66% to 99%) proved to be immunopositive. A relation to the histologic types, clinical staging, and survival was not found. Therefore, the high immunoreactivity against these oncoproteins in the same tumor cells suggests that these two oncogenes may be involved since the early stage of carcinogenesis. Loss of function of the p53 protein combined with bcl-2 upregulation might give the tumor cells a double growth advantage, because uncontrolled proliferation is combined with a reduced cell death rate. The interaction with other oncogenes may then trigger a multistep process able to promote tumor progression. The low labeling index Ki-67 was detected in nine out of 21 cases (42%), with a low percentage of tumor cells (from 3% to 15%) being positive, whereas the remaining 12 cases were negative. We found no relation to the histologic types, clinical staging, and survival; however, the low proliferation rate could explain the natural course of tumor.

Patients with multiple myeloma (MM) have increased bone marrow (BM) angiogenesis; however, the proangiogenic properties of myeloma cells and the mechanisms of MM-induced angiogenesis are not completely clarified. The angiopoietin system has been identified as critical in the regulation of vessel formation. In this study we have demonstrated that myeloma cells express several proangiogenic factors, and, in particular, we found that angiopoietin-1 (Ang-1), but not its antagonist Ang-2, was expressed by several human myeloma cell lines (HMCLs) at the mRNA and the protein levels. In a transwell coculture system, we observed that myeloma cells up-regulated the Ang-1 receptor Tie2 in human BM endothelial cells. Moreover, in an experimental model of angiogenesis, the conditioned medium of HMCLs significantly stimulated vessel formation compared with control or vascular endothelial growth factor (VEGF) treatment. The presence of anti-Tie2 blocking antibody completely blunted the proangiogenic effect of XG-6. Finally, our in vitro results were supported by the in vivo finding of Ang-1, but not Ang-2, mRNA and protein expression in purified MM cells obtained from approximately 47% of patients and by high BM angiogenesis in patients with MM positive for Ang-1, suggesting that the angiopoietin system could be involved, at least in part, in MM-induced angiogenesis.

This review summarizes the events ruled by CD38 shaping the bone marrow environment, recapitulating old and new aspects derived from the body of knowledge on the molecule. The disease models considered were myeloma and chronic lymphocytic leukemia (CLL). CD38 has been analyzed considering its twin function as receptor and enzyme, roles usually not considered in clinics, where it is used as a routine marker. Another aspect pertaining basic science concerns the role of the molecule as a member of an ectoenzyme network, potentially metabolizing soluble factors not yet analyzed (e.g., NAD+, ATP, NAM) or influencing hormone secretion (e.g., oxytocin). The last point is focused on the use of CD38 as a target of an antibody-mediated therapeutic approach in myeloma and CLL. A recent observation is that CD38 may run an escape circuit leading to the production of adenosine. The generation of local anergy may be blocked by using anti-CD38 antibodies. Consequently, not only might CD38 be a prime target for mAb-mediated therapy, but its functional block may contribute to general improvement in cancer immunotherapy and outcomes.

International audience; Glycoligands, sugar-based molecules able to complex metal cations, constitute a new class of molecules with great potential for biological and biochemical applications. To analyze their behaviour in a biological environment, we have synthesized an intrinsically fluorescent glycoligand and analyzed its trafficking in both living (U937 human cancer cells) and artificial (giant unilamellar vesicles) cell systems. We have found that this ligand has moderate cytotoxicity accompanied by specific accumulation in both living and reconstituted membranes, which it can cross to reach inner compartments.

Basic fibroblast growth factor (bFGF) is a growth factor with proangiogenetic properties. Elevated bone marrow (BM) and peripheral serum bFGF levels have been reported in patients with multiple myeloma (MM)[1][1]-[3][2]; however, the source of bFGF in patients with MM is not completely elucidated.

Hypoxia-inducible transcription factor-1 (HIF-1α) is overexpressed in multiple myeloma (MM) cells within the hypoxic microenvironment. Herein, we explored the effect of persistent HIF-1α inhibition by a lentivirus short hairpin RNA pool on MM cell growth either in vitro or in vivo and on the transcriptional and pro-angiogenic profiles of MM cells. HIF-1α suppression did not have a significant impact on MM cell proliferation and survival in vitro although, increased the antiproliferative effect of lenalidomide. On the other hand, we found that HIF-1α inhibition in MM cells downregulates the pro-angiogenic genes VEGF, IL8, IL10, CCL2, CCL5 and MMP9. Pro-osteoclastogenic cytokines were also inhibited, such as IL-7 and CCL3/MIP-1α. The effect of HIF-1α inhibition was assessed in vivo in nonobese diabetic/severe combined immunodeficiency mice both in a subcutaneous and an intratibial MM model. HIF-1α inhibition caused a dramatic reduction in the weight and volume of the tumor burden in both mouse models. Moreover, a significant reduction of the number of vessels and vascular endothelial growth factors (VEGFs) immunostaining was observed. Finally, in the intratibial experiments, HIF-1α inhibition significantly blocked bone destruction. Overall, our data indicate that HIF-1α suppression in MM cells significantly blocks MM-induced angiogenesis and reduces MM tumor burden and bone destruction in vivo, supporting HIF-1α as a potential therapeutic target in MM.

Multiple myeloma (MM) is characterized by the impaired osteogenic differentiation of human mesenchymal stromal cells (hMSCs). Canonical Wnt signaling is critical for the regulation of bone formation, however, recent evidence suggests that the non-canonical Wnt agonist Wnt5a stimulates human osteoblastogenesis through its co-receptor Ror2. The effects of MM cells on non-canonical Wnt signaling and the effect of the activation of this pathway on MM-induced osteoblast exhaustion are not known and were investigated in this study. We found that the osteogenic differentiation of bone marrow hMSCs toward osteoprogenitor cells (PreOB) significantly increased Ror2 expression, and that MM cells inhibit Ror2 expression by PreOB in co-culture by inhibiting the non-canonical Wnt5a signaling. The activation of the non-canonical Wnt pathway in hMSCs by means of Wnt5a treatment and the overexpression of Wnt5 or Ror2 by lentiviral vectors increased the osteogenic differentiation of hMSCs and blunted the inhibitory effect of MM in co-culture. Consistently, Wnt5a inhibition by specific small interfering RNA reduced the hMSC expression of osteogenic markers. Our findings demonstrate that the Wnt5a/Ror2 pathway is involved in the pathophysiology of MM-induced bone disease and that the activation of the non-canonical Wnt5a/Ror2 pathway in hMSCs increases osteogenic differentiation and may counterbalance the inhibitory effect of MM cells.

The involvement of osteocytes in multiple myeloma (MM)-induced osteoclast (OCL) formation and bone lesions is still unknown. Osteocytes regulate bone remodelling at least partially, as a result of their cell death triggering OCL recruitment. In this study, we found that the number of viable osteocytes was significantly smaller in MM patients than in healthy controls, and negatively correlated with the number of OCLs. Moreover, the MM patients with bone lesions had a significantly smaller number of viable osteocytes than those without, partly because of increased apoptosis. These findings were further confirmed by ultrastructural in vitro analyses of human preosteocyte cells cocultured with MM cells, which showed that MM cells increased preosteocyte death and apoptosis. A micro-array analysis showed that MM cells affect the transcriptional profiles of preosteocytes by upregulating the production of osteoclastogenic cytokines such as interleukin (IL)-11, and increasing their pro-osteoclastogenic properties. Finally, the osteocyte expression of IL-11 was higher in the MM patients with than in those without bone lesions. Our data suggest that MM patients are characterized by a reduced number of viable osteocytes related to the presence of bone lesions, and that this is involved in MM-induced OCL formation.

The deregulation of the homeobox genes as homeoboxB (HOXB)-7 has been previously associated to tumor progression and angiogenesis; here we investigated the potential role of HOXB7 in the pro-angiogenic properties of multiple myeloma (MM) cells. We found that HOXB7 was expressed in 10 out of 22 MM patients analyzed at the diagnosis related to high bone marrow angiogenesis and overexpressed in about 40% of myeloma cell lines compared with normal plasma cells. Enforced HOXB7 expression in MM cells by a lentiviral vector significantly modified their transcriptional and angiogenic profile, checked by combined microarray and angiogenesis PCR analyses, upregulating VEGFA, FGF2, MMP2, WNT5a and PDGFA and downregulating thrombospoindin-2. The pro- and anti-angiogenic HOXB7-related gene signature was also validated in a large independent dataset of MM patients. Accordingly, MM-induced vessel formation was significantly increased by HOXB7 overexpression both in vitro angiogenic and chorioallantoic membrane assays, as well as the HOXB7 silencing by small interfering RNA inhibited the production of angiogenic factors, and the pro-angiogenic properties of MM cells. Finally, in SCID-NOD mice we confirmed that HOXB7 overexpression by MM cells stimulated tumor growth, increased MM-associated angiogenesis and the expression of pro-angiogenic genes by microarray analysis supporting the critical role of HOXB7 in the angiogenic switch in MM.

BackgroundProstate cancer (CaP) is one of the most relevant causes of cancer death in Western Countries. Although detection of CaP at early curable stage is highly desirable, actual screening methods present limitations and new molecular approaches are needed. Gene expression analysis increases our knowledge about the biology of CaP and may render novel molecular tools, but the identification of accurate biomarkers for reliable molecular diagnosis is a real challenge. We describe here the diagnostic power of a novel 8-genes signature: ornithine decarboxylase (ODC), ornithine decarboxylase antizyme (OAZ), adenosylmethionine decarboxylase (AdoMetDC), spermidine/spermine N(1)-acetyltransferase (SSAT), histone H3 (H3), growth arrest specific gene (GAS1), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and Clusterin (CLU) in tumour detection/classification of human CaP.Methodology/principal findingsThe 8-gene signature was detected by retrotranscription real-time quantitative PCR (RT-qPCR) in frozen prostate surgical specimens obtained from 41 patients diagnosed with CaP and recommended to undergo radical prostatectomy (RP). No therapy was given to patients at any time before RP. The bio-bank used for the study consisted of 66 specimens: 44 were benign-CaP paired from the same patient. Thirty-five were classified as benign and 31 as CaP after final pathological examination. Only molecular data were used for classification of specimens. The Nearest Neighbour (NN) classifier was used in order to discriminate CaP from benign tissue. Validation of final results was obtained with 10-fold cross-validation procedure. CaP versus benign specimens were discriminated with (80+/-5)% accuracy, (81+/-6)% sensitivity and (78+/-7)% specificity. The method also correctly classified 71% of patients with Gleason score or =7, an important predictor of final outcome.Conclusions/significanceThe method showed high sensitivity in a collection of specimens in which a significant portion of the total (13/31, equal to 42%) was considered CaP on the basis of having less than 15% of cancer cells. This result supports the notion of the "cancer field effect", in which transformed cells extend beyond morphologically evident tumour. The molecular diagnosis method here described is objective and less subjected to human error. Although further confirmations are needed, this method poses the potential to enhance conventional diagnosis.

The expression of the chemokine CC-chemokine ligand 20 (CCL20)/macrophage inflammatory protein (MIP)-3α and its receptor CC-chemokine receptor 6 (CCR6) by multiple myeloma (MM) and microenvironment cells and their potential relationship with osteoclast (OC) formation and osteolytic bone lesions in MM patients was investigated in this study. First, we found that MM cells rarely produce CCL20/MIP-3α but up-regulate its production by bone marrow (BM) osteoprogenitor cells and osteoblasts in coculture with the involvement of soluble factors as interleukin-1β and tumor necrosis factor α. MM cells also stimulate both CCL20/MIP-3α and CCR6 expression by OCs in coculture. Thereafter, we showed that CCL20/MIP-3α significantly increases both the number of multinucleated tartrate-resistant acid phosphatase–positive OCs and receptor activator of nuclear factor-κB–positive OC progenitor cells similar to CCL3/MIP-1α. Finally, we found that blocking anti-CCL20/MIP-3α and anti-CCR6 antibodies significantly inhibits MM-induced OC formation. In vitro data were further expanded in vivo analyzing a total number of 64 MM patients. Significantly higher CCL20/MIP-3α levels were detected in MM patients versus monoclonal gammopathy of uncertain significance (MGUS) subjects and in MM osteolytic patients versus nonosteolytic ones. Moreover, a significant increase of CCL20/MIP-3α–positive osteoblasts in osteolytic MM patients compared with nonosteolytic ones was observed. Interestingly, no significant difference in BM CCL20/MIP-3α expression and level was observed between MGUS and nonosteolytic MM patients. Our data indicate that CCL20/MIP-3α and its receptor CCR6 are up-regulated in the bone microenvironment by MM cells and contribute to OC formation and osteolytic bone lesions in MM patients. [Cancer Res 2008;68(16):6840–50]

Angiogenesis has a critical role in the pathophysiology of multiple myeloma (MM); however, the molecular mechanisms underlying this process are not completely elucidated. The new tumor-suppressor gene inhibitor of growth family member 4 (ING4) has been recently implicated in solid tumors as a repressor of angiogenesis. In this study, we found that ING4 expression in MM cells was correlated with the expression of the proangiogenic molecules interleukin-8 (IL-8) and osteopontin (OPN). Moreover, we demonstrate that ING4 suppression in MM cells up-regulated IL-8 and OPN, increasing the hypoxia inducible factor-1α (HIF-1α) activity and its target gene NIP-3 expression in hypoxic condition. In turn, we show that the inhibition of HIF-1α by siRNA suppressed IL-8 and OPN production by MM cells under hypoxia. A direct interaction between ING4 and the HIF prolyl hydroxylase 2 (HPH-2) was also demonstrated. Finally, we show that ING4 suppression in MM cells significantly increased vessel formation in vitro, blunted by blocking IL-8 or OPN. These in vitro observations were confirmed in vivo by finding that MM patients with high IL-8 production and microvascular density (MVD) have significantly lower ING4 levels compared with those with low IL-8 and MVD. Our data indicate that ING4 exerts an inhibitory effect on the production of proangiogenic molecules and consequently on MM-induced angiogenesis.

The proteasome inhibitor bortezomib may increase osteoblast-related markers in multiple myeloma (MM) patients; however, its potential osteoblastic stimulatory effect is not known. In this study, we show that bortezomib significantly induced a stimulatory effect on osteoblast markers in human mesenchymal cells without affecting the number of osteoblast progenitors in bone marrow cultures or the viability of mature osteoblasts. Consistently we found that bortezomib significantly increased the transcription factor Runx2/Cbfa1 activity in human osteoblast progenitors and osteoblasts without affecting nuclear and cytoplasmatic active β-catenin levels. Consequently a stimulatory effect of bortezomib on bone nodule formation was also demonstrated in osteoblast progenitors. These in vitro observations were confirmed in vivo by the finding of a significant increase in the number of osteoblastic cells × mm2 of bone tissue and in the number of Runx2/Cbfa1-positive osteoblastic cells that was observed in MM patients who responded to bortezomib. Our in vitro and in vivo observations support the hypothesis that a direct stimulatory effect on bone formation process could occur during bortezomib treatment.

The chemokine receptor CXCR3, involved in chemotaxis, is expressed on normal and malignant B cells and plasma cells. Recent data suggest that CXCR3-binding chemokines may also regulate proliferation and survival in endothelial cells through the interaction with two distinct isoforms of CXCR3 (CXCR3-A and CXCR3-B).We evaluated the potential expression of CXCR3 isoforms in myeloma cells, also investigating whether CXCR3 expression is affected by cell cycle and apoptosis. Furthermore, we assessed the effect of CXCR3 activation on myeloma cell proliferation and survival.We found that CXCR3 is widely expressed on human myeloma cell lines and freshly purified myeloma cells. The presence of both CXCR3 isoforms, CXCR3-A and CXCR3-B, was observed in myeloma cells with different ratios of expression. Interestingly, we found that CXCR3 expression in myeloma cell was cell cycle dependent and that myeloma growth factors inhibited CXCR3 expression in myeloma cells. On the other hand, we found that FAS (CD95)-mediated apoptosis up-regulated CXCR3 expression. A similar behavior was observed for the CXCR3-binding chemokines. Finally we found that the activation of CXCR3 on myeloma cells by CXCL10/IP-10 partially blunted FAS-mediated apoptosis in myeloma cells that express CXCR3-A and that high concentrations of CXCL10/IP-10 inhibit myeloma cell proliferation. INTERPRETATION AND CONCLUSIONS Our data indicate that myeloma cells express the CXCR3 system with patterns correlated to cell cycle and apoptosis and that CXCR3 activation may affect myeloma cell survival and proliferation.

Bacterial antigens, such as intestinal microflora, are known to play a role in the pathogenesis of human inflammatory bowel disease (IBD). Tylosin, a macrolide antimicrobial agent, has proven to be effective in cat and dog chronic colitis, but the reasons underlying this efficacy are still unclear. In the present study we evaluated the effects of tylosin on 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis in the rat, in comparison with the antibacterial drug metronidazole and the corticosteroid budesonide. Colitis was induced by a single intrarectal administration of 10 mg TNBS under light ether anesthesia. Tylosin (20 mg/kg twice a day), metronidazole (160 mg/kg twice a day) and budesonide (500 µg/kg once a day) were given orally for up to 6 days to separate groups of rats. The animals were sacrificed after 6 days and colonic lesions evaluated (colon weight, macroscopic and histologic damage, myeloperoxidase activity). Tylosin and metronidazole significantly lowered macroscopic lesion score, reduced colon weight, the severity of histologic lesions and myeloperoxidase activity; budesonide did not significantly change the parameters of colonic inflammation. These data indicate a protective effect of tylosin against intestinal inflammation, suggesting a major role for bacteria, anaerobes in particular, in the development of TNBS-induced mucosal damage.

Hepatitis C-associated osteosclerosis (HCAO) is an impressive example of acquired diffuse osteosclerosis in adults, recently described in ten patients infected with hepatitis C virus (HCV). Its hallmark is a painful and generalized increase of bone mass. Bone biopsies show enhanced accretion rate, usually without histological abnormalities. The HCAO pathogenesis is hitherto unknown. HCV might induce a slow bone cell infection and the production of bone growth factors, such as insulin-like growth factors. Recently, receptor activator of nuclear factor-kappaB (RANK), its ligand (RANKL), and soluble decoy receptor osteoprotegerin (OPG) have been identified as a pivotal cytokine system in the bone remodeling control. We describe the 11th case of HCAO. Notably, the patient's bone biopsy showed the presence of a high number of OPG-positive osteoblasts, a slight increase of RANKL-positive stromal cells, and a dramatic reduction of the osteoclasts. Moreover, OPG serum levels were increased. These findings reported here for the first time are consistent with a pathogenetic role of the OPG/RANKL system imbalance in HCAO.

The biologic mechanisms involved in the pathogenesis of multiple myeloma (MM) bone disease are not completely understood. Recent evidence suggests that T cells may regulate bone resorption through the cross-talk between the critical osteoclastogenetic factor, receptor activator of nuclear factor-kappaB ligand (RANKL), and interferon gamma (IFN-gamma) that strongly suppresses osteoclastogenesis. Using a coculture transwell system we found that human myeloma cell lines (HMCLs) increased the expression and secretion of RANKL in activated T lymphocytes and similarly purified MM cells stimulated RANKL production in autologous T lymphocytes. In addition, either anti-interleukin 6 (anti-IL-6) or anti-IL-7 antibody inhibited HMCL-induced RANKL overexpression. Consistently, we demonstrated that HMCLs and fresh MM cells express IL-7 mRNA and secrete IL-7 in the presence of IL-6 and that bone marrow (BM) IL-7 levels were significantly higher in patients with MM. Moreover, we found that the release of IFN-gamma by T lymphocytes was reduced in presence of both HMCLs and purified MM cells. Furthermore, in a stromal cell-free system, osteoclastogenesis was stimulated by conditioned medium of T cells cocultured with HMCLs and inhibited by recombinant human osteoprotegerin (OPG; 100 ng/mL to 1 microg/mL). Finally, RANKL mRNA was up-regulated in BM T lymphocytes of MM patients with severe osteolytic lesions, suggesting that T cells could be involved at least in part in MM-induced osteolysis through the RANKL overexpression.

Although osteolysis is a common complication in patients with multiple myeloma (MM), the biologic mechanisms involved in the pathogenesis of MM-induced bone disease are poorly understood. Two factors produced by stromal-osteoblastic cells seem critical to the regulation of bone resorption: osteoprotegerin (OPG) and its ligand (OPGL). OPGL stimulates osteoclast differentiation and activity, whereas OPG inhibits these processes. The present study investigated whether myeloma cells affect physiologic OPG/OPGL balance in the bone marrow (BM) environment. Ten human myeloma cell lines and myeloma cells isolated from 26 consecutive patients with MM failed to express OPGL and only rarely produced a low amount of OPG. In a coculture system, human myeloma cells up-regulated OPGL expression but strongly down-regulated OPG production in preosteoblastic (preOB) or stromal cells (BMSCs) of primary human BM at the mRNA and protein levels. This effect, which was dependent on cell-to-cell contact between myeloma cells and BMSCs or preOB, partially involved the integrin VLA-4. In addition, overexpression of OPGL mRNA occurred in ex vivo BM cultures obtained from MM patients as compared with healthy donors, and immunohistochemical staining performed on BM biopsy specimens showed an increase of OPGL and a reduction of OPG expression in MM patients as compared with healthy subjects. In summary, these data indicate that myeloma cells affect the OPG/OPGL ratio in the BM environment and tend to confirm that the OPG/OPGL system is involved in the pathogenesis of MM-induced bone disease.

Abstract 2947 It has been previously reported that bone marrow (BM) microenvironment is hypoxic in multiple myeloma (MM) patients and that hypoxia inducible factor (HIF)-1α is overexpressed by MM cells. However, the potential role of HIF-1α as a therapeutic target in MM is not known and is currently under investigation. In this study we explored the effect of persistent HIF-1α inhibition by expression of a lentivirus shRNA pool in MM cells on proliferation, survival and transcriptional and pro-angiogenic profiles of MM cells either in vitro or in vivo in mouse models. A HIF-1α Lentivirus shRNA pool was used for HIF-1α stable knock-down in human myeloma cell lines (HMCL)s and the pKLO.1 lentiviral vector was used as the empty control vector. HMCLs were infected and then selected with puromycin. Selected clones were screened for HIF-1α, HIF-1β, HIF-2α and HIF-3α. The transcriptional profiles were evaluated in the HMCL JJN3 cells transduced with shRNA forHIF-1α (JJN3-anti-HIF-1α) and on those infected with the control vector pKLO.1 (JJN3-pKLO.1) by U133 Plus2.0 Arrays (Affymetrix®) either in hypoxic or normoxic conditions. Microarray data were further validated by quantitative real time PCR and by ELISA assays for protein levels. Finally the effect of HIF-1α inhibition in MM cells was assessed in vivo in NOD/SCID mice both in subcutaneous and intratibial models. Together with tumor volume and weight, microvascular density was evaluated by CD34 immunostaining. Cortical bone thickness was determined by microQcT in the intratibial mouse model. Among the genes significantly modulated by HIF-1α inhibition (327 and 361 genes in hypoxic and normoxic condition, respectively), we found that the pro-angiogenic molecules VEGF, IL8, IL10, CCL2, CCL5, MMP9 were down-regulated by HIF-1α inhibition. Interestingly some pro-osteoclastogenic cytokines were also inhibited including IL-7 and CCL3/MIP-1α. In the in vivo mouse models, we found that mice, injected either subcutaneously or intratibially with JJN3-anti-HIF-1α, showed a dramatic reduction in the weight and volume of the tumor burden compared to mice inoculated with the JJN3-pKLO.1. A significant reduction in the number of vessels X field and VEGF immunostaining were observed in both mouse models. Moreover in the intratibial experiments HIF-1α inhibition significantly blocked MM-induced bone destruction. Overall our data indicate that HIF-1α suppression in myeloma cells significantly blocks MM-induced angiogenesis and reduces the MM tumor burden and bone destruction in vivo suggesting that HIF-1α is a potential therapeutic target in MM. Disclosures: No relevant conflicts of interest to declare.

Abstract 3928 Multiple Myeloma (MM) is characterized by the impairment of osteogenic differentiation of bone marrow (BM) mesenchymal stromal cells (BMSC) and osteoblast suppression. Canonical Wnt signal pathway is critical in the regulation of bone formation. However recent evidences suggest that non-canonical Wnt activation by Wnt5a, rather than canonical one by Wnt3a stimulates the osteogenic properties of BMSC. Non-canonical Wnt signaling, mainly activated by Wnt5a, is transduced through FZD receptor (FDZ5) and Ror2 co-receptor to several cascades such as Disheveled pathways involving the Rho family small GTPases or the Ca++ dependent pathways/PKC involving the nuclear factor of activated T cells (NFATc). Actually, the effect of MM cells on non-canonical Wnt signaling and the role of the activation of this pathway on MM-induced osteoblastic exhaustion are still unknown and have been investigated in this study. First we checked the expression of non-canonical Wnt related molecules by BMSC and osteoprogenitor cells (PreOB) and found that both cells expressed Wnt5a but lack of express Wnt1, Wnt3a and Wnt8. The presence of the Wnt5a receptors FZD2 and FZD5 was also detected in both cell types as well as of Ror2. Interestingly we found that osteogenic differentiation of BMSC towards preOB significantly increased Ror2 and FZD5 expression. Secondly, we performed a series of co-culture between PreOB and MM cells using either IL-6 dependent (XG-1), and independent (JJN3) human myeloma cell lines or purified primary CD138+ MM cells. We found that XG-1 and CD138+ MM cells inhibit Ror2 and FZD5 expression in PreOB and consistently the activity of NFATc1 at nuclear level. Thereafter the activation of non-canonical WNT pathway in PreOB, checked by the intracytoplasmatic increase of Ca++influx, phospho-PKC expression and NFATc1 activity, was induced either by Wnt5a treatment or by Wnt5a overexpression through a lentivirus vector. Ror2 overexpression was also performed by lentivirus vector in PreOB. The transcriptional profiles of both PreOB overexpressing Wnt5a and Ror2 have been evaluated by GeneChip® HG-U133Plus 2.0 arrays. The raw intensity signals were extracted from CEL files and normalized using the RMA package for Bioconductor and custom GeneAnnot-based Chip Definition Files in R software. We found that Wnt5a treatment as well as Wnt5a overexpression significantly increased osteogenic differentiation and the expression of the osteogenic markers alkaline phosphatase and collagen I in PreOB. Consistently with these observations, we also demonstrated that siRNA-mediated Wnt5a silencing inhibited these osteogenic markers in the same cell type. Moreover we found that the activation of non-canonical WNT signal pathway in PreOB, blunted the inhibitory effect of MM cells on the osteogenic differentiation process in co-culture. Finally, we show that Ror2 overespression in PreOB activated non-canonical Wnt signaling, increased osteogenic differentiation and restored the osteogenic properties of PreOB in co-culture with MM cells. In conclusion, our data indicate that activation of non-canonical Wnt5a/Ror2 pathway in BM osteoprogenitor cells increases osteogenic differentiation and counterbalances the inhibitory effect of MM cells suggesting that modulation of Wnt5/Ror2 pathway in the microenvironment could be a target for MM bone disease. Disclosures: Bolzoni: Celgene Italy: Research Funding. Giuliani:Celgene: Research Funding; Novartis: Research Funding.

Abstract 3927 Hypoxia-inducible factor (HIF)-1α is a critical trigger and regulator of tumor associated angiogenesis. It has been previously reported that bone marrow (BM) microenvironment is hypoxic in multiple myeloma (MM) patients and that HIF-1α is overexpressed by CD138+ MM cells and modulates the transcriptional and pro-angiogenic profiles of MM cells. The potential role of HIF-1α as a therapeutic target in MM is under investigation. To deepen this issue, in this study we explored the effect of a stable HIF-1α inhibition in MM cells on cell proliferation, survival and on MM-induced angiogenesis either in vitro or in vivo using a plasmocytoma model. Human myeloma cell lines (HMCL)s were screened for HIF-1α protein expression in normoxia condition finding that 2 out 5 HMCLs were positive (JJN3 and RPMI-8226). Anti-HIF-1α Lentivirus sh RNA pool was used for HIF-1α stable knock-out in human myeloma cell lines (HMCL)s whereas the pKLO.1 lentiviral vector was used as the empty control vector. HMCLs have been infected and selected with 0.4 ug/ml puromycin for 21 days and the selected clones screened for HIF-1α, HIF-1β, HIF-2α and HIF-3α. A selective inhibition on HIF-1α mRNA and protein expression was observed both in normoxic and hypoxic conditions. HMCLs proliferation and apoptosis were not affected by HIF-1α inhibition neither in hypoxia nor normoxia. Thereafter the transcriptional profiles on the HMCL JJN3 transduced with shRNA anti-HIF-1α (JJN3-anti-HIF-1α), as compared to those infected with the control vector pKLO.1 (JJN3-pKLO.1) have been analyzed by U133 Plus2.0 Arrays (Affymetrix ®) either in hypoxic or normoxic conditions. The raw intensity signals were extracted from CEL files and normalized using the RMA package for Bioconductor and custom GeneAnnot-based Chip Definition Files in R software. Absolute fold change higher than 1.5 was used to select differentially expressed genes. Among the genes significantly modulated (327 and 361 genes in hypoxic and normoxic condition, respectively) we found that the pro-angiogenic molecules VEGF, IL8, IL10, CCL2, CCL5, MMP9 were down-regulated in JJN3-anti-HIF-1α as compared to JJN3-pKLO.1 either in hypoxic or normoxic conditions. Microarray data were further validated by quantitative real time PCR. In line with this observation, we found that the pro-angiogenic properties of JJN3, evaluated by an in vitro angiogenesis assay (Angio-Kit, TCS), were significantly inhibited by the HIF-1α knock-out. Finally the potential involvement of HIF-1α in MM-induced angiogenesis was assessed in vivo in NOD/SCID mice in a subcutaneous model. We found that the mice which were injected subcutaneously with JJN3-anti-HIF-1α, showed a dramatic reduction of the weight and volume of the plasmocytoma mass than mice inoculated with the JJN3-pKLO.1 (P=0.0007 and P=0.012; respectively). Moreover we observed a significant reduction of the number of vessels X field stained by CD31 antigen (anti-HIF-1α vs. pKLO.1: −76%; P=0.003) and VEGF immunostaining, whereas the Ki67 index of the plasmocytoma mass only showed a slight reduction (anti-HIF-1α vs. pKLO.1: −14%, P=NS). Overall our data indicate that HIF-1α suppression in myeloma cells significantly blocks MM-induced angiogenic switch both in vitro and in vivo and consequently reduces the MM tumoral burden suggesting that HIF-1α is a potential therapeutic target in MM. Disclosures: Bolzoni: Celgene Italy: Research Funding. Giuliani:Celgene: Research Funding; Novartis: Research Funding.

Abstract 422 Multiple myeloma (MM) is characterized by the accumulation of malignant plasmacells into the bone marrow (BM) microenvironment that supports their growth and survival. Particularly, an increase of BM angiogenesis occurs in relationship with plasmacells infiltration playing a critical role in the progression on monoclonal gammopathy. Hypoxia is known to be associated to angiogenesis in solid tumors as well as the hypoxia-inducible factor (HIF)-1α is a critical trigger and regulator of the angiogenic switch. Actually, the oxygen levels in the BM of patients with monoclonal gammopathy as well the effects of hypoxia and HIF-1α on the gene expression and pro-angiogenic profile of CD138+ cells are not known. In this study, first, we investigated the level of BM oxygen saturation (sO2) and partial pressure (pO2) in a cohort of 44 patients with monoclonal gammopathy at the diagnosis including active MM (n°=25), smoldering MM (n° = 8) and MGUS (n°=11) showing that BM of MM patients was hypoxic (mean ± SD of pO2: 52.5 ± 8.69 mmHg; sO2 of 83.7 ± 11%) even if any significant difference in both pO2 and sO2 was not observed in comparison with smoldering MM patients or MGUS as well as in relationship with the stage of disease (p=0.7). Next, we evaluated how hypoxia exposition could modify the gene expression profile of CD138+cells isolated form MM patients by U133 Plus2.0 Arrays. By supervised analysis performed on 11 paired samples we found that hypoxia significantly modulated 714 genes in CD138+ cells isolated from MM patients. Interestingly, genes belonging either to oxidative stress and hypoxia signaling, including heme oxygenase 1 (HMOX1) and the heat shock proteins 90kDa alpha (HSP90AA1; HSP90AB1), or to protein ubiquitination pathway, such as XIAP, were significantly up-regulated by hypoxia. Among the pro-angiogenic genes, we found that both VEGFA and IL8 were induced by hypoxia. Following, given that HIF-1α accumulates only in hypoxic condition, we checked HIF-1α protein expression in the BM of MM patients by immunohistochemistry on bone biopsies immediately after fixation. A strong HIF-1α immunostaining was demonstrated in MM cells at nuclear level in all patients analyzed. Interestingly, the presence of HIF-1α protein was also observed in isolated CD138+ MM cells of about 28% of MM patients in normoxic condition. In order to investigate the potential role of HIF-1α on MM cell gene expression and angiogenic profile we performed HIF-1α silencing in MM cells by siRNA anti-HIF-1α and that exposed cells to normoxic or hypoxic conditions. HIF-1α suppression was associated to the modulation of pro-angiogenic molecules (VEGFA, VEGFB, IL-8 and PGF) and oxidative stress (SOD2, PTGS2, TXT) and glycogenolysis regulating (ENO2, ALDOC, PFKFB3, HK2, PDK1, PFKFB4) genes. Data obtained were than validated by real time PCR and at protein level by western blot and ELISA assay. Consistently we found that HIF-1α suppression significantly inhibited the pro-angiogenic properties, evaluated in an in vitro angiogenesis model as capillary junctions and tubules formation and tubule length. In conclusion, we demonstrate that the MM-BM environment is hypoxic and that HIF-1α protein expressed by MM cells. Consistently we show that hypoxia and HIF-1α significantly modulate the gene expression profiles of MM cells regulating the expression of the pro-angiogenic factors by MM cells and their pro-angiogenic properties. Disclosures: No relevant conflicts of interest to declare.

Abstract 125 It is known that bone marrow (BM) angiogenesis is increased in multiple myeloma (MM) patients in relationship with disease progression and supports MM cell growth. MM-induced angiogenesis is mainly due to an overproduction of pro-angiogenic molecules by MM cells and the BM microenvironment. However, the molecular mechanisms at the basis of the angiogenic process are currently under investigation. The deregulation of the homeobox genes has been previously associated to tumor progression and neoangiogenesis. Particularly, overexpression of the homeobox HOXB7 is involved in tumor-associated angiogenic switch in solid tumors. Interestingly, we have recently shown that HOXB7 is one of the overexpressed genes in BM micorenvironment cells in MM patients as compared to healthy subjects. In this study we have investigated the expression of HOXB7 by MM cells and its potential role in MM-induced angiogenesis in vitro and in vivo. First, by microarray analysis we evaluated HOXB7 expression in MM cells finding that HOXB7 was overexpressed in 43% of 23 human myeloma cell lines (HMCLs) tested as compared to normal plasmacells. HOXB7 expression was further confirmed by real time PCR and western blot analysis. On the other hand a small fraction of patients (about 10% in a representative database of 133 MM patients) showed HOXB7 overexpression with a preferential distribution based on the characteristics of molecular MM subtypes being significantly overexpressed in those cases which did not show any of the primary IGH translocations, corresponding to the TC2 and TC3 groups in the translocations/cyclins (TC) classification. In order to investigate the potential role of HOXB7 in MM cells either we enforced HOXB7 expression by lentivirus vectors in JJN3 HMCL (JJN3-HOXB7) or we silenced its expression by appropriate siRNA in the KMS20 HMCL that constitutively over-expressed HOXB7. Interestingly we found that cell proliferation was significant higher in JJN3-HOXB7 as compared to JJN3 transduced with the empty vector (JJN3-pWPI) as well as HOXB7 downregulation in KMS20 significantly reduced their cell proliferation and viability. Following we evaluated the whole transcriptional and specifically the pro-angiogenic profile of JJN3-HOXB7 as compared to JJN3-pWPI using Genechips® HG-U133Plus 2.0 and an angiogenesis oligonucleotide arrays. We found that the pro-angiogenic cytokines as VEGFA, FGF2, MMP2 and PDGFA were significantly upregulated whereas we identified the specific downregulation of the angiogenic inhibitor TSP2 in JJN3-HOXB7. Data were then validated at protein level by ELISA assay in cell-conditioned media (CM). Consistently, we found that CM of JJN3-HOXB7 significantly stimulated vessel formation as compared to JJN3-pWPI using both in vitro angiogenic model (Angiokit) and chorioallantoic membrane assay. In line with these observations, we found that HOXB7 silencing by siRNA in KMS20 reduced the expression of pro-angiogenic molecules, including VEGF and FGF2, and growth factors. Finally the potential role of HOXB7 in MM-induced angiogenesis was tested in vivo in NOD/SCID mice. We found that mice injected with JJN3-HOXB7 cells developed a significantly bigger tumor mass than mice inoculated with the JJN3-pWPI (p=0.0039) with higher number of vessels stained by CD31 antigen. Moreover, the transcriptional and angiogenic profiles of the tumor mass indicated the upregulation of VEGFA, FGF2, MMP2, PDGFA and WNT5a as well as the downregulation of TSP2, at both mRNA and protein level in mice colonized by JJN3-HOXB7 as compared to those inoculated with JJN3-pWPI, confirming in vitro observations. In conclusion our data demonstrate the critical role of HOXB7 in the production of pro-angiogenic molecules by MM cells and in the regulation of MM-induced angiogenic switch suggesting that HOXB7 could be a potential therapeutic target in MM. Disclosures: No relevant conflicts of interest to declare.

Hypoxia is a common feature of solid tumors associated to angiogenesis and malignant phenotype. Tumor adaptation to hypoxia is mainly due to the hypoxia-inducible factor (HIF)-1α, a key transcription factor that regulates angiogenesis and tumor progression. As known multiple myeloma (MM) is a hematological malignancy characterized by the accumulation of malignant plasmacells into a hypoxic microenvironment as the bone marrow (BM) that critically supports their growth and survival. However the effect of hypoxia on MM cells and the role of HIF-1α in MM-induced angiogenesis actually are not known. Recently we have demonstrated that the tumor suppressor gene ING4 may exert an anti-angiogenic effect through the inhibition of HIF-1α activity in MM cells in hypoxic condition suggesting a role of HIF-1α in MM-induced angiogenic switch. To go further insight this issue, in this study, first we checked the level of BM oxygen tension in a cohort of MM patients (n°=25) at the diagnosis as compared to healthy donors and MGUS subjects. The mean pO2 ± SD was 52.3±9 mmHg (p=NS) in MM patients similar to that observed in the controls, confirming that MM cells are exposed in vivo to hypoxic microenvironment. Thereafter HIF-1α protein expression by MM cells was checked by immunohistochemistry on bone biopsies showing the presence of HIF-1α stabilization at nuclear level in malignant plasmacells as well as in BM stromal cells (BMSC) into the BM. Consequently the effect of hypoxia and HIF-1α in both MM and BMSC cells was checked. Human myeloma cell lines (JJN3 and RPMI-8226) and BMSC were transfected with a pool of siRNA anti-HIF-1α to knockout HIF-1α and then exposed to low oxygen tension. A gene expression profiling evaluation was performed by microarray analysis using Gene Chips U133plus 2.0 (Affymetrix). Data were then validated by real time PCR. We found that hypoxia significantly upregulated the expression of the pro-angiogenic molecules in both MM and BMSC cells including Vascular Endothelial Growth Factor (VEGF), Osteopontin (OPN) and Interleukin-8 (IL-8) blunted by siRNA anti-HIF-1α. Genes belonging to glycolysis and HIF-1α regulating signal pathways were found to be also regulated by HIF-1α in MM cells in hypoxic condition. These observations were confirmed in purified CD138+ MM cells (n°=11) exposed to hypoxia that induced a significant up-regulation of the pro-angiogenic molecules and the modulation of glycolysis and ubiquitin mediated proteolysis signal pathways. Finally, the potential expression and role of HIF-1α in MM cells was also investigated in normoxic condition. Whereas the presence of HIF-1α mRNA was observed in all HMCLs and primary MM cells tested, HIF-1a protein stabilization and activity was observed at nuclear level in 2 out of 6 HMCLs and in about 38% of MM patients evaluated suggesting that a hypoxia independent stabilization of HIF-1α may occur in MM cells. Consistently, in normoxic condition, HIF-1α knock out by siRNA significantly affected in HMCLs either pro-angiogenic molecules as VEGF or several genes belonging to cell cycle regulation. In conclusion our data underline the role of hypoxia in the regulation of the angiogenic signature of MM cells and the BM microenvironment and suggest that HIF-1α could be a potential target in MM.

Osteoclast (OC) activation in multiple myeloma (MM) is primarily due to the imbalance of the critical osteoclastogenic system RANKL/OPG in the bone microenvironment. Recent evidences indicate that chemokines, small chemoattractant proteins involved in cancer cell homing, may contribute to osteoclast formation and activation. However, whereas the role of the chemokine macrophage inflammatory protein (MIP)-1α in MM-induced OC activation is well established, the involvement of other chemokines is not known. In this study, we evaluated the potential role of MIP-3α/CCL20 and its receptor CCR6 in the pathophysiology of OC formation and osteolytic lesions in MM. First the effect of MIP-3α/CCL20 on in vitro osteoclast formation by peripheral monocytes was evaluated. (MIP)-3α/CCL20 significantly increased both the number of multinucleated TRAP+ OCs and RANK+ OC progenitor cells in presence of RANKL. In addition we found that (MIP)-3α/CCL20 increases RANKL mRNA levels in both human osteoblastic (OB) and bone marrow (BM) osteoprogenitor cells (preOB). Following, the potential production of (MIP)-3α/CCL20 by human MM cell lines (HMCLs) and fresh purified CD138+ MM cells was also checked. Significant levels of (MIP)-3α/CCL20 were detected in one out of nine HMCLs tested and in about 10% of purified MM cells by ELISA and immunohystochemistry. On the other hand we found that MM cells up-regulated (MIP)-3α/CCL20 secretion, in OB/PreOB cells and in OCs as well as its receptor CCR6 in OCs in co-culture systems in presence of a transwell insert. Among potential soluble factors involved in the up-regulation of MIP-3α/CCL20 by MM cells we found that IL-1β and TNFα together stimulate MIP-3α/CCL20 production in both OB and PreOB. The role of MIP-3α/CCL20 in OC activation by MM cells was finally demonstrated by finding that both blocking anti-(MIP)-3α/CCL20 and anti-CCR6 Abs. but not anti-IgG control significantly decreased OC formation induced by the conditioned medium of MM cells co-cultured with OB and OC, respectively. This chemokine system was further studied in vivo in MM patients. MIP-3α/CCL20 levels were detected in the BM plasma of MGUS subjects (n°=16) and in MM (n°=52) patients at the diagnosis in relationship with the presence of bone lesions (osteolytic n°= 32; non-osteolytic: n°=20). Significant higher MIP-3α/CCL20 levels were detected in MM patients vs. MGUS (mean ± SD: 51.9±2 vs. 21±3 pg/mL; p=0.01) and in MM osteolytic patients vs. non-osteolytic ones (mean ± SD: 70.8±5.9 vs. 13.8±1.1 pg/mL; p=0.001). Interestingly, no significant differences were observed between MGUS and non-osteolytic MM patients. By immunohystochemistry performed on BM biopsies, we consistently found that MIP-3α/CCL20 was over-expressed in OBs in osteolytic MM patients as compared to non-osteolytic ones. In addition we found that OCs showed a strong CCR6 staining in the areas with an increased number of OCs. In conclusion our data indicate that (MIP)-3α/CCL20 its receptor CCR6 are up-regulated in bone microenvironment by MM cells and involved in osteoclast formation and bone lesions in MM patients.

It is well established that osteoblast formation and function are profoundly impaired in multiple myeloma (MM) patients. Osteoblastic cells also regulate myeloma cell growth and increasing bone formation result in a reduction of tumoral burden in mice. Recent data suggest that ubiquitin-proteasome pathway, the major cellular degradative system and therapeutic target in myeloma cells, also regulates osteoblast differentiation. Further it has been demonstrated that different proteasome inhibitors may stimulate bone formation in mice. Finally, preliminary observations obtained in MM patients treated with the proteasome inhibitor Bortezomib show an increase of bone specific alkaline phosphatase in responder patients as compared to non-responder ones. Currently it is not know whether the proteasome inhibitor Bortezomib may have a direct effect on osteoblast and bone formation in vitro human cultures and in vivo in MM patients. To clarify this issue first we checked the effect of Bortezomib either on osteoblast differentiation and formation or on osteoblast proliferation, survival and function. In long-term human BM cultures we found that Bortezomib did not reduce the number of both early bone marrow (BM) osteoblast progenitors Colony Forming Unit-Fibroblast (CFU-F) and late ones Colony Forming Bone nodules (CFU-OB). On the other hand we found that Bortezomib (2–3 nM) significantly induced osteoblast phenotype in human mesenchymal cells incubated in presence of osteogenic factors. A stimulatory effect on osteoblast markers was observed after 24 hours of Bortezomib treatment. Consistently we found that Bortezomib significantly increased the activity of the transcription factor Runx2/Cbfa1 in human osteoblast progenitors without affecting the canonical WNT signaling pathway checked by the evaluation of nuclear and cytoplasmatic active beta-catenin levels. Using the human osteoblast like cells MG-63 and immortalized normal osteoblasts (HOBIT) we found that Bortezomib at concentration ranging between 2nM and 5nM did not inhibit osteoblast proliferation or induce osteoblast apoptosis. Similarly, Bortezomib did not affect the expression of osteoblast markers, Runx2/Cbfa1 activity and WNT signaling in both MG-63 and HOBIT cells. To extent our in vitro observation we have evaluated the potential effect of Bortezomib in vivo in MM patients. Bone histomorphometry as well as immunostainig for Runx2/Cbfa1 and beta-catenin was performed on BM biopsies obtained from 15 MM patients before and after 6–8 cycles of Bortezomib administrated in mono-therapy. A significant increase in the number of osteoblastic cells X mm2 of bone tissue and in the number of Runx2/Cbfa1 positive osteoblastic cells was observed only in responder patients showing an early increase of the serum alkaline phosphatase. In conclusion our data indicate that Bortezomib may increase osteoblast differentiation in human mesenchymal cells without affecting the proliferation, survival and function of mature osteoblasts. In vivo and in vitro observations support the hypothesis that both direct and indirect effects on bone formation process could occur during Bortezomib treatment.

Osteoblast impairment occurs within myeloma (MM) cell infiltration into the bone marrow (BM). Wnt signaling is involved in the regulation of osteoblast formation. Canonical Wnt signaling pathway is activated by Wnt 1/3a that induce the activation of GSK3/Axin complex leading to the stabilization and nuclear translocation of beta-catenin that in turn activates the transcription system Lef1/TCF. Recently it has been reported that MM cells produce the Wnt inhibitors DKK-1 demonstrating a correlation between its expression and the presence of bone lesions in MM patients. However the effect of MM cells on Wnt signaling cascade in osteoblasts and osteoblast progenitors has not been investigated. To clarify this issue, first we checked DKK-1 production by human myeloma cell lines (HMCLs), purified CD138+ MM cells and BM plasma of MM patients by PCR and ELISA. Following we performed a co-culture system with HMCLs or CD138+ MM cells and either human osteoblast line (HOBIT) and with BM osteoprogenitor cells (PreOB) obtained after differentiation from mesenchymal cells or murine osteoprogenitor cell lines C2C12 and MC3T3. Both DKK-1 positive HMCLs (XG-1 and JJN3) and negative ones (RPMI-8226, OPM-2) have been used in co-culture as well as DKK-1 positive and negative purified CD138+ MM cells. Similarly we tested the effect of BM plasma of MM patients positive and negative for DKK-1 production on both human and murine cells. Wnt signaling in osteoblasts and osteoblast progenitors was evaluated either at mRNA level by specific human and murine Wnt Array kits and by quantitative PCR or at protein one by Western blot analysis for GSK3b/Axin and LEF-1/TCF expression. We evaluated active de-phosphorylated beta-catenin and inactive phosphorilated one by westernblot and by ELISA in cytosolic and nuclear extracts. DKK-1 median levels detected in the conditioned media of XG-1 and JJN3, MM cells and in BM plasma of DKK-1 positve MM patients were 0.60 ng/mL and 0.38 and 8.84 (range: 1.55–91) ng/mL respectively. Any significant inhibitory effect on WNT signaling and active beta-catenin expression and levels was not observed in HOBIT and human PreOB after co-culture with both HMCLs and MM cells or BM plasma independently to DKK-1 expression. On the contrary DKK-1 positive MM cells or BM plasma suppressed active beta-catenin expression in murine osteoprogenitor cell lines in presence of BMP-2. Consistently Wnt3a stimulation as well as anti-DKK-1 abs. did not restore the inhibitory effects on osteoblast formation and differentiation induced by MM cells in human PreOB. Consistently any significant difference was not detected on beta-catenin expression by stromal/osteoblastic cells on bone biopsies by immunohistochemistry between osteolytic (n°=10) and non-osteolytic (N°=10) MM patients. The different behavior between human and murine osteoblastic cells was further investigated. We found that both cells expressed significant levels of active beta-catenin however DKK-1 suppressed active nuclear and cytosol beta-catenin at concentration of 20–30 ng/mL in C2C12 and MC3T3 whereas only DKK-1 concentrations higher to 500 ng/mL are able to inhibited beta-catenin in HOBIT and human PreOB as well as osteoblast formation and differentiation in human BM cultures. In conclusion our data indicate that MM cells block canonical Wnt signaling in murine osteoblastic cells but not in human osteoblasts and osteoblast progenitors. Beta-catenin independent mechanisms could be involved in DKK-1 mediated bone destruction in MM patients.

A recent experimental and clinical observation on the use of bone marrow-derived stem/progenitor cells (BM-SC) to treat non-hematologic degenerative diseases has renovated the long-standing knowledge of their biology and function. However, the paradoxical lack of histological studies limits the identification of SC within the human BM, constraining their purification and further obscuring the sites where SC fate is decided. In an attempt to identify human hematopoietic BM-SC and their relationships with the tissue environment, bone marrow biopsies from 31 subjects without BM involvement by hemo-lymphoproliferative diseases were analysed with stringent morphometric criteria for the quantification of cells expressing CD117 (c-kit) and CD34, as candidate SC markers, and/or CD45, CD3, CD20, myeloperoxidase (MPOX), CD68, Glycophorin A (GlyA) and Factor VIII (vWF), as lineage commitment. Immunofluorescence combined with confocal examination was also performed to allow the simultaneous detection of multiple surface/cytoplasmic antigens together with transcription factors. Hematopoietic cells constituted 54% of the tissue among which MPOX, GlyA and vWF positive cells represented 43.8%, 13.5% and 0.06% of marrow cells, respectively. The frequency of CD34pos cells was 93.5±43.6 each 105 total nucleated cells; c-kit involved 2.7±2.6/105 cells and c-kit-CD34 co-expression was present in 1.14±0.98/105cells. When the incidence of CD45 was evaluated among these two cell populations only 15% of c-kitpos and 49% of CD34pos cells showed hematopoietic lineage commitment, indicating the presence in the human bone marrow of undifferentiated SC. The analysis of the expression of Terminal Deoxynucleotidyl Transferase (TdT) in hematopoietic cells demonstrated that 10% of c-kitpos, 20% of CD34pos and 0.62% of CD45pos cells possessed the nuclear transcription factor. The distribution of BM-SC was predominantly scattered throughout the tissue although interstitial and paratrabecular clusters were detected and are currently under intense scrutiny in search of BM-SC sites of activation. In a subset of patients, the histological analysis was complemented by the cytofluorimetric estimation of the same BM cells populations. The results of this study may contribute to the improvement in the isolation and characterization of BM-SC aimed to the identification of their niches and to an appropriate clinical application in the field of regenerative medicine.

Multiple Myeloma (MM) cells grow into the bone marrow (BM) mainly through the interactions with the microenvironment. The marked induction of angiogenesis by myeloma cells suggests that this process has a pivotal role in the progression of MM. However the molecular mechanisms underlying the regulation of angiogenesis remain unresolved. The new candidate tumor suppressor gene “inhibitor of growth family member 4” (p29ING4) has been recently implicated in solid tumors as a repressor of angiogenesis and tumor growth through the association with p65 subunit of NF-kB and the suppression of angiogenic related gene as IL-8 (Garkavtsev I et al., Nature 2004). In this study first we have evaluated the mRNA expression of p29ING4 in human myeloma cell lines (HMCLs): U266, RPMI-8226, XG-1, XG-6, OPM-2 and JJN3, in EBV+ lines HS-SULTAN and ARH-77 and in purified CD138+ plasmacells (purity >90%) obtained from either 32 MM patients or 6 patients with MGUS by both qualitative RT-PCR and quantitative real time PCR. Healthy CD20+ BM B lymphocytes and/or CD138+ plasmacells obtained from reactive tonsils have been used as controls. p29ING4 has been checked at protein level by western blot on nuclear extracts using a polyclonal anti-p29ING4 Ab. In addition, p29ING4 mRNA levels have been compared with IL-8, VEGF, Ang-1 mRNA levels and correlated with BM angiogenesis in MM patients. The presence of p29ING4 transcript was observed in HMCLs even if p29ING4 mRNA levels were significantly reduced in all HMCLs tested as compared to controls with a median ΔCt p29ING4 (Ct p29ING4-Ct Abl ): 3.2 vs. 0.45 (p 25 pg/ml) have a significantly lower p29ING4 mRNA levels (median ΔCt p29ING4:4.71 vs. 1.9; p=0.04). Finally we found that MM patients with high microvalscular density (MVD) and number of vessels X field have significant lower p29ING4 mRNA levels as compared to those with low MVD (median ΔCt p29ING4:4,8 vs. 2; p=0.04). Our data indicate that the tumor suppressor p29ING4 is down-regulated in myeloma cells and regulate their pro-angiogenic properties.

Bone marrow (BM) angiogenesis is increased in Multiple Myeloma (MM) patients and correlates with disease progression and patient survival. Myeloma cells secrete the main endothelial growth factor VEGF. In mouse models VEGF secretion as well as the angiogenic properties of MM cells correlate with the lack of CD45 expression by MM cells. However, recent data indicate that VEGF plasma cell expression is similar between MGUS and MM patients suggesting that other molecules could be involved. In line with this hypothesis we have recently demonstrated that myeloma cells may also produce factors with angiogenic properties as angiopoietin-1 (ANG-1) and osteopontin (OPN) that are involved in myeloma induced angiogenesis in vitro. In order to identify which factors correlate with BM angiogenesis in MM patients, we have investigated in a cohort of 121 newly diagnosed MM patients (stage I–III) the expression of the angiogenic molecules VEGF, ANG-1 and OPN and their correlation with bone marrow (BM) angiogenesis and CD45 expression by MM cells. We found that 90% of CD138+ MM cells tested were positive for VEGF mRNA. On the other hand we found that 50% and 40 % of MM patients were positive for ANG-1 and OPN mRNA respectively. Using the previously published cut off for CD45 expression we found that 61 out of 121 MM patients were positive for CD45 and 60 out of 121 were negative for CD45 expression. Any correlation was not observed between VEGF expression and BM angiogenesis in MM patients (p=0.5), whereas the number of microvessels X field was higher in Ang-1 positive patients in comparison with Ang-1 negative ones (mean±SE: 6.23±0.2 vs. 2.94±0.1, median: 6.21 vs. 2.79; p=0.001,) and the microvascular density (MVD) was significantly increased (32.98±1.7 vs. 14.55±1.3, median: 34.69 vs. 13.04; p Our data indicate that ANG-1 and in part OPN rather than VEGF and CD45 expression by MM cells are the critical determinants correlated with the increase of BM angiogenesis that occurs in MM patients at the diagnosis.

Abstract 131 Multiple myeloma (MM)–induced osteolysis is characterized by severely imbalanced and uncoupled bone remodeling due to increased osteoclast recruitment and suppressed osteoblast differentiation. Osteocytes are located in the lacuna/canalicular system of bone and that have been recently hypothesized to regulate local bone remodeling through the cell death and apoptosis triggering osteoclast recruitment and formation. Actually, the potential involvement of osteocytes in MM-induced osteoclast formation and in the bone remodeling alterations occurring in MM patients is still unknown and was investigated in this study. Firstly we evaluated the effect of MM cells on osteocyte survival in a co-culture transwell system of human myeloma cell lines (JJN3, KMS12, XG-1) and the human pre-osteocytic cell line HOB-01. By Transmission Electron Microscopy (TEM) observations and TUNEL assay we showed the occurrence of either apoptotic cells or degenerated non-apoptotic cells in the monolayer of HOB-01 cells co-cultured with MM cells or treated with their conditioned media (CM) as compared to non-treated cells suggesting that MM cells may increase osteocyte cell death. Second, we investigated whether MM cells could affect the pro-osteoclastogenic profile and the osteoclastogenic properties of HOB-01 in co-culture. The microarray analysis, using Affymetrix GeneChip® HG-U133Plus2.0 platform) identified 47 genes significantly modulated by MM cells in HOB-01, most of them up-regulated; among these, we identified the increased expression of the pro-osteoclastogenic genes IL11 and MMP1, a metalloproteinase involved in tumoral ostelysis. The up-regulation of both IL-11 and MMP-1 was then confirmed at both mRNA and protein level either by real time PCR and ELISA assay, respectively. Furthermore, the mRNA expression and the protein levels of the main pro-osteoclastogenic cytokines CCL3/MIP-1α and RANKL were also measured in the co-cultures showing that CCL3/MIP-1α levels were significantly higher than those observed either in HOB-01 or MM cells cultured alone whereas RANKL levels and RANKL/OPG ratio were unchanged. Analyzing separately HOB-01 and MM cells after the co-culture period, we demonstrated that increased levels of CCL3/MIP-1α were due to its up-regulation in MM cells. In line with the increase of the pro-osteoclastogenic cytokines observed in the co-cultures, we found that the CM of HOB-01 co-cultured with MM cells significantly increased CD14+-derived osteoclastogenesis in presence of RANKL as compared to the CM of HOB-01 or MM cells cultured alone. Interestingly this effect was completely blunted in presence of blocking antibody anti-CCL3/MIP-1α and in a lesser extent of anti-IL-11 and anti-MMP-1. To translate into a clinical perspective such in vitro evidences, then we performed histological analysis on bone biopsies obtained from iliac crest of a cohort of 32 patients with MM at the diagnosis or relapsed (ISS I-III, mean age±SD: 72±10), 55% of them with osteolytic bone lesions and 10 patients with monoclonal gammopathy of uncertain significance (MGUS) (mean age± SD: 71±14). Ten sex-age matched healthy subjects without hematological malignancies or metabolic bone disease were also analyzed. Consistently with our in vitro observations, we found a significant reduction in the percentage of viable osteocytes with an increase of the number of death osteocytes/empty lacunae in MM patients as compared to healthy subjects (p=0.003) but not MGUS (p=0.4). Under Light Microscopy and TUNEL analysis we demonstrated that osteocyte cell death in MM patients was due, at least in part, to an increase of the osteocyte apoptosis. As regard the skeletal involvement in MM patients we showed significant lower percentage of viable osteocytes (p=0.05) and higher amount of death osteocytes and empty lacunae in osteolytic vs. non-osteolytic MM patients (p=0.05). Finally a significant negative correlation was observed in MM patients between the amount of viable osteocytes and that of osteoclasts [Spearman (r): -0.33, p=0.04]. Overall our data suggest a critical role of osteocytes in MM-induced osteolysis in MM patients showing that the interaction of MM cells with osteocytes trigger osteoclastogenesis through the increase of osteocyte apoptosis and the production of pro-osteoclastogenic cytokines. Disclosures: No relevant conflicts of interest to declare.