The aim of this study was to investigate the role of Notch signaling in intrinsic and bone marrow stromal cells (BMSC)-mediated drug resistance in multiple myeloma (MM) and in MM-stem cell (SC) niche maintenance. MM is an incurable hematological malignancy due to intrinsic or BMSC-mediated drug resistance; the hyperexpression of two Notch ligands, Jag1 and 2 in MM increases Notch signaling in MM cells and BMSCs resulting in malignant cells survival and proliferation. Notch pathway supports stem cell maintenance and drug resistance is an intrinsic feature of cancer stem cells; MM stem cells (MM-SCs) have been characterized as CD138- subpopulation. MM-SCs are resistant to common drugs used in therapy and responsible for disease relapse. MM cell lines were cultured alone or co-cultured with NIH3T3 murine fibroblasts or HS5 human BMSC line. To detect apoptosis induced by Mitoxantrone, Bortezomib and Melphalan, AnnexinV+ cells were processed by flow cytometry (FC). Jag1 and Jag2 were transiently silenced in MM cells using specific siRNAs. The gene expression levels were analyzed by quantitative RT-PCR. Anti-apoptotic proteins were assessed by FC. Notch inhibition was obtained by g-secretase inhibitor and the effect on MM cell stemness potential was assessed by FC measure of CD138- MM cells or clonogenic serial replating in methylcellulose-based medium. Our results demonstrate that Jag1 and 2 silencing reduces anti-apoptotic genes expression, i.e. SDF1α, CXCR4, Bcl-XL, Bcl2, Survivin and ABCC1 and increases sensitivity of MM cells to the used drugs. MM cells and BMSCs reciprocally activate Notch signaling resulting in increased drug resistance due to: i) an elevated expression of the anti-apoptotic genes in MM cells; ii) BMSCs release of soluble factors, i.e. SDF1α and VEGF, relevant for MM cell growth and survival. Interestingly, Jag1 and 2 silencing in MM cells co-cultured with BMSCs could reverse all gene and protein expression changes as well as BMSCs protective effect increasing the apoptotic rate of MM cells. In addition, we show in MM cell lines that DAPT-mediated Notch inhibition decreases MM-SCs and reduces the clonogenic ability in serial replating. The evidence that Jag1 and 2 silencing affects the intrinsic and BMSC-induced drug resistance in MM cells also by affecting the MM-SC population supports the rationale for a Notch-tailored approach to overcome the unavoidable relapse pf MM patient.

The role of notch pathway in multiple myeloma associated drug resistance / S. Garavelli, E. Lazzari, M. Colombo, N. Platonova, M.T. Palano, F. Baccianti, S. Galletti, A. Neri, L..A. Crews, C.H. Jamieson, R. Chiaramonte. - In: CANCER RESEARCH. - ISSN 0008-5472. - 77:13 suppl.(2017 Apr), pp. 1-1. ((Intervento presentato al convegno American association for cancer research annual meeting tenutosi a Washington nel 2017 [10.1158/1538-7445.AM2017-LB-025].

The role of notch pathway in multiple myeloma associated drug resistance

S. Garavelli;E. Lazzari;M. Colombo;N. Platonova;PALANO, MARIA TERESA;S. Galletti;A. Neri;R. Chiaramonte
2017-04

Abstract

The aim of this study was to investigate the role of Notch signaling in intrinsic and bone marrow stromal cells (BMSC)-mediated drug resistance in multiple myeloma (MM) and in MM-stem cell (SC) niche maintenance. MM is an incurable hematological malignancy due to intrinsic or BMSC-mediated drug resistance; the hyperexpression of two Notch ligands, Jag1 and 2 in MM increases Notch signaling in MM cells and BMSCs resulting in malignant cells survival and proliferation. Notch pathway supports stem cell maintenance and drug resistance is an intrinsic feature of cancer stem cells; MM stem cells (MM-SCs) have been characterized as CD138- subpopulation. MM-SCs are resistant to common drugs used in therapy and responsible for disease relapse. MM cell lines were cultured alone or co-cultured with NIH3T3 murine fibroblasts or HS5 human BMSC line. To detect apoptosis induced by Mitoxantrone, Bortezomib and Melphalan, AnnexinV+ cells were processed by flow cytometry (FC). Jag1 and Jag2 were transiently silenced in MM cells using specific siRNAs. The gene expression levels were analyzed by quantitative RT-PCR. Anti-apoptotic proteins were assessed by FC. Notch inhibition was obtained by g-secretase inhibitor and the effect on MM cell stemness potential was assessed by FC measure of CD138- MM cells or clonogenic serial replating in methylcellulose-based medium. Our results demonstrate that Jag1 and 2 silencing reduces anti-apoptotic genes expression, i.e. SDF1α, CXCR4, Bcl-XL, Bcl2, Survivin and ABCC1 and increases sensitivity of MM cells to the used drugs. MM cells and BMSCs reciprocally activate Notch signaling resulting in increased drug resistance due to: i) an elevated expression of the anti-apoptotic genes in MM cells; ii) BMSCs release of soluble factors, i.e. SDF1α and VEGF, relevant for MM cell growth and survival. Interestingly, Jag1 and 2 silencing in MM cells co-cultured with BMSCs could reverse all gene and protein expression changes as well as BMSCs protective effect increasing the apoptotic rate of MM cells. In addition, we show in MM cell lines that DAPT-mediated Notch inhibition decreases MM-SCs and reduces the clonogenic ability in serial replating. The evidence that Jag1 and 2 silencing affects the intrinsic and BMSC-induced drug resistance in MM cells also by affecting the MM-SC population supports the rationale for a Notch-tailored approach to overcome the unavoidable relapse pf MM patient.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2434/508122
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