Unknown,Transcriptomics,Genomics,Proteomics

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Lenalidomide and Pomalidomide inhibit Multiple Myeloma-induced osteoclast formation and RANKL/OPG ratio in myeloma microenvironment targeting the expression of adhesion molecules.

ABSTRACT: Multiple myeloma (MM)-induced osteoclast (OC) formation occurs in close contact with MM cell infiltration into the bone marrow (BM) due to the imbalance of the receptor activator of NF-kappa-B ligand (RANKL)/osteoprotegerin (OPG) ratio in favor of RANKL in the micorenvironment. Soluble factors including CCL3/MIP-1?, IL7 and IL-3 also contribute to the increased OC formation in MM.The immunomodulatory drugs (IMiDÂ®s) directly inhibit OCs, however their effect on the mechanisms involved in MM-induced OC formation are not known and have been investigated in this study. We found that both Lenalidomide (LEN) and Pomalidomide (POM), at concentration ranging reached in vivo, significantly blunted RANKL up-regulation normalizing the RANKL/OPG ratio in human BM osteoprogenitor cells (PreOBs) co-cultured with MM cells and inhibited CCL3/MIP-1? production by MM cells. The reduction of CD49d expression on MM cells, a molecule critically involved in RANKL up-regulation in the micorenvironment, accompanied this effect. Consistently the pro-osteoclastogenic property of the conditioned medium of MM cells co-cultured with PreOBs was reduced in the presence of both IMiDÂ®s. By microarray analysis we further investigated the effect of POM and LEN on the transcriptional profile of both MM cells and PreOBs. We found a significant down-regulation in MM cells, in addition to CD49d, of genes belonging to the adhesion molecules family such as ITGA8 and ICAM2 (CD102) induced by both IMiDÂ®s compounds. In conclusion our data suggest that POM and LEN inhibits MM-induced OC formation through the inhibition of RANKL/OPG ratio targeting the expression of adhesion molecules by MM cells. This series of microarray experiments contains the gene expression profiles of JJN3 cell line cultured in presence of POM (100 ÂµM), LEN (100 ÂµM) or vehicle in triplicate flasks for 24 hours, respectively. Preparation of biotin-labeled cRNA, hybridization to GeneChipÂ® Human Genome U133 Plus 2.0 Arrays (Affymetrix Inc., Santa Clara, CA, USA) and scanning of the chips (7G Scanner, Affymetrix Inc.) were carried out according to manufacturer's protocols. Expression values were extracted from cell intensity files, using GeneAnnot custom chip definition files. The CDF Packages for R/Bioconductor (http://www.bioconductor.org/) version 2.2.0 was based on GeneAnnot annotation Version 2.0 and publicly available at http://www.xlab.unimo.it/GA_CDF/. The data were normalized using Robust Multi-array Average procedure.

ORGANISM(S): Homo sapiens

SUBMITTER: Antonino Neri

PROVIDER: E-GEOD-37302 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:In multiple myeloma (MM), hypoxia-inducible transcription factor-1 (HIF-1) is overexpressed in the MM cells of the hypoxic bone marrow (BM) microenvironment. Herein, we explored in MM cells the in vitro and in vivo effects of persistent HIF-1 inhibition by expression of a lentivirus shRNA pool on proliferation, survival and transcriptional and pro-angiogenic profiles. Among the significantly modulated genes (326 and 361 genes in hypoxic and normoxic condition, respectively), we found that HIF-1 inhibition in the human myeloma cell line JJN3 downregulates the pro-angiogenic molecules VEGF, IL8, IL10, CCL2, CCL5, and MMP9. Interestingly, several pro-osteoclastogenic cytokines were also inhibited, such as IL-7 and CCL3/MIP-1. The effect of HIF-1 inhibition was assessed in vivo in NOD/SCID mice both in subcutaneous and intratibial models, indicating in either case a dramatic reduction of weight and volume of the tumor burden as a consequence of HIF-1 knockdown. Moreover, a significant reduction of the number of vessels per field and VEGF immunostaining were observed. Finally, in the intra-tibial experiments, HIF-1 inhibition significantly blocks JJN3-induced bone destruction. Overall, our data indicate that HIF-1 suppression in MM cells significantly blocks MM-induced angiogenesis and reduces both tumor burden and bone destruction in vivo, strongly indicating HIF-1 as an emerging therapeutic target in MM. The transcriptional profiles on JJN3 transduced with shRNA anti-HIF-1 (JJN3-anti-HIF-1), as compared to those infected with the control vector pLKO.1 (JJN3-pLKO.1), have been analyzed either in hypoxic or normoxic conditions. To perform gene expression profiles, total RNA was purified using the RNeasy Total RNA Isolation Kit (Qiagen, Valencia, CA). Preparation of biotin-labeled cRNA, hybridization to GeneChip Human Genome U133 Plus 2.0 Arrays and scanning (GeneChip¨ Scanner 3000 7G, Affymetrix Inc.) were performed according to manufacturer's protocols.

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Lenalidomide and Pomalidomide inhibit Multiple Myeloma-induced osteoclast formation and RANKL/OPG ratio in myeloma microenvironment targeting the expression of adhesion molecules.

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