ALBERT

Description: 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.

Description: Osteopontin (OPN) is a multifunctional bone matrix glycoprotein that it is involved in angiogenesis, cell survival and tumor progression. OPN gene expression by human cells is mainly regulated by the bone specific trascription factor Runx2 namely also CBFA1 (Runx2/Cbfa1) even if other transcription factors may be also involved in the production of OPN by cancer cells as the CCAAT/enhancer-binding protein alpha (C/EBPα) and AML-1. In this study we show that human myeloma cell lines (HMCLs): RPMI-8226, U266, XG-1 and XG-6 but not ARH-77 and normal CD19+ cells directly produce OPN and express its major regulating gene Runx2/Cbfa1. The activity of Runx2/Cbfa1 in human myeloma cells has been also demonstrated by a gel mobility shift assay. On the other hand we found that all HMCLs tested were negative for C/EBPα mRNA, whereas AML-1A and AML-1B mRNA were expressed in all HMCLs even if any correlation has not been found between OPN expression and AML-1A/AML-1B ratio. In addition we found that OPN production by myeloma was up-regulated at both mRNA and protein level by IL-6 and IGF-I through a Runx2/Cbfa1 mediated mechanism; in turn recombinant human OPN was able to increase myeloma cells proliferation. The potential role of OPN in MM-induced angiogenesis has been also investigated in an experimental model of angiogenesis. rhOPN treatment stimulated vessel formation as compared to control and the conditioned medium (CM) of HMCLs, significantly increased vessel formation in comparison either with control or with VEGF treatment. On the contrary OPN-immunodepleted CM of HMCLs had not a stimulatory effect on vessel formation and the presence of anti OPN Ab inhibited vessel formation induced by HMCLs. The expression of OPN by purified bone marrow (BM) CD138+ cells has been also 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 control subjects. A significant higher MVD was observed in the group of patients positive for OPN, (mean±SE: 29.1±0.7 vs. 17.55±0.37; p

Description: 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.

Description: Multiple Myeloma (MM) cells are extremely resistant to apoptosis and currently new potential drug combinations are under investigation. We have shown that the combined treatment with the MEK1/2 inhibitor PD184352 (PD) and Arsenic Trioxide (ATO) resulted in the synergistic (Combination Index

Description: Recently, it has been demonstrated that Wnt inhibitors, as DKK-1 and sFRP-3 are produced by MM cells. High DKK-1 levels in MM cells have been shown to correlate with the presence of focal bone lesions in MM patients. However no data are available concerning the potential relationship between sFRP-3 and DKK-1 expression by MM cells, their BM levels and the presence of bone disease. In this study we have investigated DKK-1 and sFRP-3 mRNA expression by RT-PCR in fresh purified CD138+ MM cells (purity〉95%) obtained from 50 newly diagnosed MM patients and 16 MGUS. The expression of both molecules was also evaluated by microarray analysis (Affimetrix U133A chips) in an independent larger database of 102 newly diagnosed MM patients. DKK-1 and sFRP-3 protein expression in MM cell lysates was detected by western blot analysis. In addition DKK-1 and sFRP-3 levels were detected by ELISA into the bone marrow (BM) plasma. Bone status in all MM patients tested was evaluated by total X-rays scan. By RT-PCR we found that 74% of MM patients were positive for DKK-1 mRNA at the diagnosis whereas 60% expressed sFRP-3 mRNA. DKK-1 and sFRP-3 expression was further analyzed in relationship with the bone status of MM patients. Although DKK1 and sFRP-3 were found positive in 81% and 73% of osteolytic patients, and 50% and 37% of non-osteolytic ones respectively, these values do not reach a statistical significance (Chi Square 2-tailed p=0.2) and 36% sFRP-3 (p=0.069). On the other hand, comparing MM patients positive for DKK-1 and sFRP-3 with those negative for both molecules we found a significant statistical correlation with the presence of bone lesions (p=0.03). In agreement with RT-PCR analysis, supervised gene expression profiling of 102 MM patients failed to find a significant correlation between the level of DKK-1 and sFRP-3 mRNA expression and the presence of bone lesions in MM patients. When we analyzed DKK-1 and sFRP-3 protein expression in malignant purified plasma cells we found that a lower number of MM patients expressed Wnt inhibitors as compared to mRNA. On the other hand, DKK-1 and sFRP-3 were was detectable in bone marrow (BM) plasma of 90% and 76% of of MM patients, respectively. Significant higher DKK-1 and sFRP-3 levels were detected in MM patients (median DKK-1 levels: 2.84 ng/mL, range:0,55–91,55 ng/mL; sFRP-3: 1,53 ng/mL, range: 0–27 ng/mL) as compared to MGUS subjects (DKK-1: 1,5 ng/ml, range: 0–4,12 ng/mL; sFRP-3: 0,55 ng/mL, range: 0–6,82 ng/mL) (Nonparametric 2-tailed test. p=0.005 and p=0.003, respectively). Considering the bone status of MM patients we found that osteolytic patients showed significant higher BM DKK-1 levels as compared to non-osteolytic ones (median: 6,85 vs 1,19 ng/m; p= 0.05) whereas BM sFRP-3 ones did not reach a statistical significance (3,3 vs. 1,12 ng/mL p=0.3). In conclusion, our results support the hypothesis that Wnt inhibitors DKK1 and s-FRP-3 may have a role in bone disease in MM indicating that DKK-1 and sFRP-3 double positive MM patients may have higher incidence of bone lesions. Finally, our study indicate that BM levels DKK-1 rather than the level of mRNA expression by MM cells better correlated with the presence of bone lesions in MM patients suggesting the involvement of the BM microevironment as source of DKK-1.

Description: Studies of gene expression performed on multiple myeloma (MM) cells have leaded to identify molecules able to inhibit osteoblast differentiation. Whereas potential alterations occurring in the bone microevironment cells in MM patients are not completely elucidated. To clarify this issue we have developed a method to direct isolate mesenchymal cells (MSC) and osteoblastic cells (OB) without in vitro differentiation from trabecular bone biopsies obtained by iliac crest of MM patients (n°=24) with or without osteolytic bone lesions. Bone status was evaluated in all MM patients by total X rays scan and NMR for the spine. MSC and OB isolated from trabecular bone of healthy donors underwent to orthopedics surgery was used as controls. Cell proliferation in relationship with growth substrate (bone and glass) was evaluated in isolated MSC (osteolytic n°=9, non-osteolityc n° 15) and OB cells (osteolytic n°=9, non-osteolityc n°=11) as well as immunophenotype by FACS analysis, protein pattern by immunohistochemical staining and ELISA assay and finally gene expression profiling by microarray (Affimetrix). First the presence of potential contaminating cells was excluded by FACS analysis in all the samples tested being both MCS and OB obtained negative for CD3, CD14, CD20 and CD138 antigens. We found that cell proliferation was significantly higher in MSC as compared to OB in MM patients and that both MSC and OB cells have higher cell doubling rate as compared to controls. Immunophenotype reveals a different pattern of expression of the chemokine receptors CXCR4, CXCR5, CCR6 in MSC and OB in the different group of patients. Higher alkaline phosphatase (AP) expression was observed in OB versus MSC in non-osteolitic patients but not in osteolityc ones. In line with these observations we found that the expression of the osteoblast transcrition factor Runx2/CBFA1 was higher in MSC obtained from non-osteolytic patients as compared with osteolytic ones. Hierarchical clustering by unsupervised analysis of gene expression profiles (Affymetrix U133A chips) identified two major cluster branches containing respectively MSC and OB cells, with subgroups correlated with the bone status. Following supervised analysis, a total of 121 probe-set were found differentially expressed in MSCs from patients with/without osteolytic lesions (57 up-regulated and 64 down-regulated) Distinct patterns of gene expression profiling were observed in MSCs versus Obs when osteolityc or non-osteolytic patients were compared. Interestingly, markers and transcription factors known to be specific for osteoblast cells were up-regulated in OB versus MSC in non-osteolytic patients but not in osteolytic ones. Notably, a significant downregulation of Runx2 and AP-1 related pathways was observed in OB of osteolytic MM patients as compared to non-osteolytic ones. In conclusion in this study for the first time we have identified a different pattern of growth, phenotype and gene expression in isolated MSC and OB cells in relationship with the bone status of MM patients highlighting the critical role of the block of osteoblast differentiation and the involvement of the related signature pathways.

Description: 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.

Description: Bone destruction in multiple myeloma is characterized both by markedly increased osteoclastic bone destruction and severely impaired osteoblast activity. We reported that interleukin-3 (IL-3) levels are increased in bone marrow plasma of myeloma patients compared with healthy controls and that IL-3 stimulates osteoclast formation. However, the effects of IL-3 on osteoblasts are unknown. Therefore, to determine if IL-3 inhibits osteoblast growth and differentiation, we treated primary mouse and human marrow stromal cells with IL-3 and assessed osteoblast differentiation. IL-3 inhibited basal and bone morphogenic protein-2 (BMP-2)-stimulated osteoblast formation in a dose-dependent manner without affecting cell growth. Importantly, marrow plasma from patients with high IL-3 levels inhibited osteoblast differentiation, which could be blocked by anti-IL-3. However, IL-3 did not inhibit osteoblast differentiation of osteoblastlike cell lines. In contrast, IL-3 increased the number of CD45+ hematopoietic cells in stromal-cell cultures. Depletion of the CD45+ cells abolished the inhibitory effects of IL-3 on osteoblasts, and reconstitution of the cultures with CD45+ cells restored the capacity of IL-3 to inhibit osteoblast differentiation. These data suggest that IL-3 plays a dual role in the bone destructive process in myeloma by both stimulating osteoclasts and indirectly inhibiting osteoblast formation. (Blood. 2005;106:1407-1414)

Description: 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)