Multiple myeloma-derived Jagged ligands increases autocrine and paracrine interleukin-6 expression in bone marrow niche

Multiple myeloma cell growth relies on intrinsic aggressiveness, due to a high karyotypic instability, or on the support from bone marrow (BM) niche.We and other groups have provided evidences that Notch signaling is related to tumor cell growth, pharmacological resistance, localization/recirculation in the BM and bone disease.This study indicates that high gene expression levels of Notch signaling members (JAG1, NOTCH2, HES5 and HES6) correlate with malignant progression or high-risk disease, and Notch signaling may participate in myeloma progression by increasing the BM levels of interleukin-6 (IL-6), a major player in myeloma cell growth and survival. Indeed, in vitro results, confirmed by correlation analysis on gene expression profiles of myeloma patients and immunohistochemical studies, demonstrated that Notch signaling controls IL-6 gene expression in those myeloma cells capable of IL-6 autonomous production as well as in surrounding BM stromal cells. In both cases Notch signaling activation may be triggered by myeloma cell-derived Jagged ligands. The evidence that Notch signaling positively controls IL-6 in the myeloma-associated BM makes this pathway a key mediator of tumor-directed reprogramming of the bone niche.This work strengthens the rationale for a novel Notch-directed therapy in multiple myeloma based on the inhibition of Jagged ligands

Hypoxia increases multiple myeloma stem cells by modulating notch signalling

BACKGROUND Multiple myeloma (MM) is a haematological tumor characterized by malignant plasma cells accumulation in the bone marrow (BM). Notch pathway receptors and ligands are upregulated in MM. Hypoxia is a low oxygen condition resulting in HIF-1a-mediated hypoxic response resulting in tumor neoangiogenisis and progression. Recent reports indicate that HIF-1a may positively regulate Notch signalling and enhance the expression of downstream genes. In MM, hypoxia can sustain MM stem cell (MM-SC) population. METHODS OPM2 and H929 MM cells underwent a hypoxia mimic treatment with 100\u3bcM Cobalt Chloride (CoCl2). HIF-1a and Notch pathway members were analyzed by Western Blot or qRT-PCR. A Notch reporter assay was performed in OPM2 upon CoCl2 treatment. MM-SCs analysis was performed by flow cytometry for CD138 and methylcellulose clonogenic assay. Notch inhibition was obtained by 25mM DAPT. RESULTS CoCl2-mediated hypoxia sustained MM-SC niche by increasing CD138neg population. Moreover, it increased HIF-1a and the Notch pathway members, Jag1 and ICN2, at protein and gene level, resulting in an increased Notch-activated transcription. DAPT-mediated Notch inhibition was able to revert hypoxia-mediated amplification of MM-SCs indicating that Notch pathway acts downstream hypoxia in MM-SC self-renewal. DISCUSSION Our results concerning hypoxia ability to amplification MM-SC population and to induce Notch pathway activation in MM cells by upregulating Notch receptors and ligands is consistent with results in other types of cancer. For the first time we demonstrate that Notch pathway is a hypoxia downstream effector in supporting the amplification of the MM-SC population . CONCLUSION Our results indicate that hypoxia can activate Notch pathway in MM and sustain MM-SC niche

Multiple myeloma associated angiogenesis: the notch pathway in the interplay between myeloma and endothelium

Multiple myeloma(MM) is a still incurable cancer that strongly depends on interactions with bone marrow(BM) microenvironment. Patients display high level of angiogenesis due to endothelial cells(ECs) malignant behavior as compared to healthy counterpart(1). Moreover, MM cells show dysregulation in Notch signaling pathway due to Jag ligands overexpression. This condition brings to the formation of a sustaining loop between MM cells with surrounding cells including bone marrow stromal cells(BMSCs) and osteoclasts. Although the role of Notch in MM progression, it is not clear its involvement in MM-EC crosstalk. Thus, this project aims to investigate this aspect of MM. Notch ligands, Jag1 and 2, were silenced in RPMI8226 cell line using short hairpin RNAs (RPMI8226shJAG1/2). For ECs, Human pulmonary aortic endothelial cells (HPAEC) was used and for BMSCs, the GFP+HS5 cell line. Matrigel and Wound Healing assays were set up to study Notch involvement in modulating angiogenesis potential of MM cells co-cultured with HPAEC and ECs motility in response to MM-produced soluble factors. To mimic MM niche, we generated an organoid exploiting a decellularized extracellular matrix as scaffold. We evaluated apoptosis of MM cells alone or in co-culture with BMSCs and with ECs by flow cytometry. Matrigel assay of HPAEC cultured with RPMI8226scrb cells showed that direct contact increased angiogenic potential of ECs; this effect was reduced in absence of Jag ligands, indicating an active role of Notch in ECs stimulation. Wound Healing assay demonstrates that the silencing of Notch signaling also influences HPAEC motility. Concerning the organoid, our results indicate that the dECM is suitable for cells seeding and 3D structure generation. Moreover, apoptosis assays show that MM cells display and increased survival in presence of BMSCs, confirming their protective role; we do not obtain significant difference in MM apoptosis in presence, or not, of ECs. Surprising, we observe that MM cells protect ECs suggesting that myeloma can improve angiogenesis also by preventing ECs apoptosis. These results indicate a novel and active role of Notch in MM-EC crosstalk; 3D organoid can mimic microenvironment and it can be used as novel tool to study BM microenvironment beyond animal models

Role of notch pathway in EV-mediated crosstalk between multiple myeloma and tumor microenvironment

INTRODUCTION Multiple myeloma (MM) is an incurable cancer characterized by a strong tropism to bone marrow (BM) and an important involvement of Notch pathway. Recently, extracellular vesicles (EVs) have been reported as mediators in creating a supportive milieu for MM. These evidences make us to hypothesize that it may have a role in EVs mediated cross-talk. EXPERIMENTAL MODEL We established two MM cell lines that stably retain the doxycycline-inducible pTRIPZ vector containing either scrambled or anti Jagged1/Jagged2 shRNA, and a bone marrow mesenchimal stromal cell (BM-MSC) line (HS5) that expresses shRNAs for Notch1 and 2 and the scrambled control. EVs were isolated by ultracentrifugation and used for functional assays. RESULTS Notch1/2 KD in BM-MSC results in a decrease in the release of EVs; BMSC-EV derived from Notch1/2 KD cells showed a reduced ability to induce Bortezomib resistance and to stimulate migration of MM cells. On the other side, MM-EVs are able to activate Notch in HS5, increasing the production of pro-tumoral factor SDF1; the effect is lost when EVs are isolated from MM cells where Jag1/2 were silenced. Moreover EVs released by co-cultures of BMSCs and MM cells silenced for Jag1/2 display a reduced ability to increase osteoclastogenesis compared with EVs from the control culture. CONCLUSION We prove that BM-MSC and MM derived EVs show biological functions in accordance with Notch pathway activation status and their role as mediators of tumor progression and relapse

Multiple myeloma-endothelium: 2D and 3D systems to study notch signaling

INTRODUCTION: Multiple myeloma (MM) is characterized by the accumulation of monoclonal plasma cells within bone marrow (BM), where they interact with surrounding BM cells. MM-associated angiogenesis is key mechanism, which correlates with disease progression. Notch pathway deregulation in MM is due to Notch receptors and Jagged ligands overexpression, has pro-tumor effects and is involved in MM crosstalk with different cell types. Here, we investigate the role of Notch pathway in the crosstalk between MM cells and endothelial cells (ECs). MATERIALS AND METHODS: RPMI8226 cells were transduced with lentiviral vectors carrying shRNAs for Jagged1 and 2 (RPMI8226-JAG1/2) or scrambled shRNAs (RPMI8226scrb). Angiogenic potential was assessed by Matrigel assay using Human Pulmonary Artery Endothelial Cells (HPAECs) and RPMI8226-JAG1/2 or RPMI8226scrb cells (ratio 1:2). 3D-co-culture was set up by loading BMSCs (HS5), MM cells (RPMI8226) and ECs (HPAEC) (ratio 1:2:1) in decellularized extracellular matrix produced by NIH3T3 cells. Apoptosis was assessed by AnnexinV staining and flow-cytometry. RESULTS: RPMI8226scrb cells display angiogenic activity lost by RPMI8226-JAG1/2. We set up 3D-organoids that that recapitulate the interaction of MM cells with key BM cellular components (ECs and mesenchymal cells) and result in angiogenesis and prevention of tumor cell apoptosis. DISCUSSION and CONLUSION: Overexpressed Jagged ligands in MM cells are key in activating Notch signaling in nearby ECs and the consequent MM-associated angiogenesis. 3D-organoids are a useful tool to study MM-associated angiogenesis and reveal that MM cells can sustain vessels formation. We are going to investigate in details the role of Notch by 2D and 3d assays

Understanding the role of extracellular vesicles in the interaction of multiple myeloma with the surrounding microenvironment: the key contribution of the Notch pathway

Introduction Multiple myeloma (MM) is an incurable cancer characterized by a strong tropism to the bone marrow (BM) and an important involvement of the Notch pathway. Recently, extracellular vesicles (EVs) have been reported as novel mediators in creating a supportive milieu for MM. Here we investigate the role of the activated Notch signaling in EVs mediated cross-talk. Materials and methods We established two MM cell lines stably retaining the doxycycline-inducible pTRIPZ vector containing anti-Jagged1 and Jagged2 shRNAs and a BM mesenchymal stromal cell (BMSC) line expressing shRNAs for Notch1 and Notch2. EVs were isolated by ultracentrifugation and used for functional assays and molecular analysis. Results We present evidences that EVs play a crucial role in the dysregulated interactions of MM cells with the BM microenvironment and that Notch regulates their release. Indeed, BMSCs knockdown for Notch1/2 results significantly reduced EVs release and their ability to induce Bortezomib resistance in MM cells and stimulate their migration. On the other side, MM-derived EVs activate Notch signaling in HS5, increasing the production of pro-tumor factors; the effect is lost when EVs are isolated from MM cells knockdown for Jagged1/2. Moreover, EVs released by co-cultures of BMSCs and MM cells silenced for Jagged1/2 display a reduced ability to increase osteoclastogenesis compared with EVs from the control culture. Discussion and Conclusions These new insights in the pathophysiology of the de-arranged BM niche represent the rationale for a therapy aiming to uncouple the crosstalk of MM with the surrounding microenvironment by inhibiting Notch signaling

Understanding the interaction between myeloma and the bone marrow niche: role of the Notch pathway in extracellular vesicles-mediated communication

Multiple myeloma (MM) is an incurable hematological tumor stemming from malignant plasma cells. MM cells accumulate in the bone marrow (BM) and establish complex interactions with normal BM stroma, which promotes tumor survival, drug resistance and the development of bone disease. The Notch oncogenic pathway provide a key contribute to the ability of MM cells to shape the BM niche, affecting both MM cell biology and the interplay with the surrounding normal cells. Recently, extracellular vesicles (EVs) have been shown to be crucial for this insidious interaction. This work investigated the effects of Notch inhibition on the EV-mediated crosstalk between neoplastic MM cells and stromal cells, providing evidences that: 1) Notch activity is essential for stroma-derived EVs to promote MM cells migration and drug resistance; 2) MM cells derived EVs are able to activate Notch in stromal cells boosting their ability to secrete pro-tumor factors; 3) Notch is fundamental for MM-derived EVs pro-osteoclastogenic potential. Taken together these results indicate that the Notch pathway promotes the EV-mediated interaction of MM cells and normal BM cells resulting in drug resistance and osteolytic activity, suggesting that a Notch\u2013tailored approach may be effective in targeting the EV-mediated pathological interplay in the BM niche

Role of extrac ellular vesicles-mediated communication in the crosstalk between myeloma cells and the bone marrow niche

BACKGROUND-AIM Multiple Myeloma (MM) is characterized by clonal proliferation of PCs in the bone marrow (BM) microenvironment. Here, the interaction between MM cells and BM promotes tumour survival, drug resistance and the development of bone disease. The Notch oncogenic pathway provides a key contribute to the ability of MM cells to shape the BM niche, affecting both MM cell biology and their ability to educate the surrounding normal cells. Recently, extracellular vesicles (EVs) have been reported as novel mediators in creating a supportive milieu for MM. Here we investigate the role of EV-mediated cross-talk in determine MM cells ability to shape the bone marrow niche, promoting the pro-tumor behavior of normal stromal cells. METHODS EVs were isolated by ultracentrifugation and used for functional assays and molecular analysis. EVs characterization was performed by NTA (Nanoparticle Tracking Analysis); qPCR was performed using SYBR Green. Uptake assay was analyzed by confocal imaging. Apoptosis analysis was performed by flow cytometry; evaluation of protein expression was achieved by flow cytometry or western blot. TRAP assay was used to assess osteoclasts differentiation. RESULTS We present evidences that EVs play a crucial role in the dysregulated interactions of MM cells with the BM microenvironment. EVs derived by BM stromal cells (BMSCs) are able to induce resistance to standard of care drugs (i.e. Bortezomib, Melphalan) and can to stimulate MM cells migration. Interestingly, the inhibition of the oncogenic Notch signaling can revert this effect. On the other side, MM-derived EVs are able to increase the production of pro-tumor factors by BMSCs (i.e. SDF1\u3b1, IL6) . Finally, EVs released by MM cells are able to carry the main osteoclastogenic factor RANKL and to boost osteoclast differentiation. Consistently, the inhibition of the Notch pathway, one of the key regulator of RANKL production, causes a decrease in MM-EVs pro-osteoclastogenic ability compared with EVs from the control culture. This is particularly relevant due to the crucial role played by bone disease in MM progression. CONCLUSION These new insights in the pathophysiology of the de-arranged BM niche represent the rationale for at therapy aiming to uncouple the crosstalk of MM with the surrounding microenvironment to hamper the progression of this disease