The CD138+ MM cell population probably also includes the side population, which is a CD138low+ subpopulation with stem cell properties.46 Matsui et al47,48 have previously reported predominant effect and activity of Hh signaling in CD138? MM stem cells, although we observed that CD138+ cells are also susceptible to inhibition by NVP-LDE225. activation of both canonical and noncanonical Hh pathway in MM, thus providing the rationale for screening Hh inhibitors in clinical trials to improve MM patient end result. Introduction The Hedgehog (Hh) pathway regulates multiple processes involved in development and differentiation of tissues and organs during embryonic life.1 Recently, it has become obvious that Hh signaling retains some activity even during adult life: in mature tissues, it regulates tissue homeostasis and repair and, in those tissues undergoing constant renewal, such as skin, colon, liver, and blood, it is also implicated in maintaining a stem/progenitor cell compartment,2,3 explaining how the Hh pathway deregulation may cause developmental defects during the embryonic life.1,4 Its abnormal activity can also lead to tumorigenesis during adult life either by stem cell pool expansion2,3 or mutations affecting the normal growth-regulatory mechanisms.5C7 An aberrant expression of developmental genes from Wnt and Hh pathways has been reported during malignant transformation of multiple myeloma (MM) cells.8 Several findings support a role of Hh signaling in regulating a stem cell niche also in MM9 and in modulating clinical response to conventional and novel therapeutic agents.10 Indeed, Hh ligands produced by murine bone marrow stromal cells (BMSCs) support growth and survival of human main CD19+ lymphoma and CD138+ MM cells, demonstrating a role Tuberculosis inhibitor 1 of the Hh pathway both in lymphoma and in terminally differentiated MM cells.11 Finally, we recently showed ciliary protein overexpression as a possible cause of constitutive and noncanonical Hh pathway activation, suggesting a cilia-dependent mode of Hh signaling in MM.12 Aberrant Hh signaling has been described in almost all tumors and is associated with 3 possible mechanisms: genetic alterations, autocrine and/or paracrine Hh activity, and option and synergistic pathways leading to gene activation.13,14 Sonic (Shh), Desert (Dhh), and Indian (Ihh) hedgehog are the ligands for the pathway. The signaling is usually brought on by binding of endogenously or exogenously produced ligand to Patched1 (Ptch1) on target cells. This prospects to inhibition of Ptch1 via cellular internalization and Smoothened (Smo) localization around the cell surface, both by a cilium-mediated mechanism.15 Smo activation prospects to nuclear translocation of Glioma (Gli) transcription factors followed by expression of Gli target genes, including and gene overexpression in CD138+ plasma cells (PCs) from persons with monoclonal gammopathy of undetermined significance (MGUS) compared with CD138+ PCs from healthy persons, MM and plasma cell leukemia (PCL) patients, suggesting that aberrant Hh activation is important in disease initiation. We further demonstrate that both canonical and Smo-dependent as well as noncanonical and Smo-independent mechanisms can contribute to Hh signaling activation in MM. Finally, we provide evidence that NVP-LDE225, a novel synthetic Smo antagonist currently in clinical development,16,17 decreases MM cell viability in vitro by inducing specific down-regulation of Gli1 and Ptch1, hallmarks of cell response to Hh pathway. Moreover, the combination of NVP-LDE225 with bortezomib in vitro has a modest additive effect compared with either drug alone, and the in vivo study shows an increased antitumor activity RGS4 by the combination compared with bortezomib alone. Methods Microarray gene expression The gene expression data were generated using our Affymetrix HG-U133A GeneChip Arrays (Affymetrix) from 4 healthy donors, 11 MGUS, 133 MM, and 9 PCL patients and from 23 human MM cell lines (HMCLs), obtained as previously explained18 and deposited in National Center for Biotechnology Information’s Gene Expression Omnibus (http://www.ncbi.nlm.mih.gov/geo) as accession no. “type”:”entrez-geo”,”attrs”:”text”:”GSE13591″,”term_id”:”13591″,”extlink”:”1″GSE13591 and “type”:”entrez-geo”,”attrs”:”text”:”GSE6205″,”term_id”:”6205″,”extlink”:”1″GSE6205, respectively. All samples were normalized and analyzed using the bioconductor function for Robust Multi-array Analysis19 in which perfect match intensities were background adjusted and normalized by means of quantile-quantile normalization. Gene expression levels were expressed as mean SEM. The statistical significance of differences among the groups was assessed using Tuberculosis inhibitor 1 Kruskal-Wallis test. values Tuberculosis inhibitor 1 .05 were considered significant. Circulation cytometry Circulation cytometry-based evaluation of Hh-protein expression was performed using a panel of MM cell lines and MM patient-derived BMSCs. U266, NCIH929, RPMI8226, and MM1R were obtained from ATCC. KMS12BM and KMS12PE were from German Collection of Microorganisms and Cell Cultures (DSMZ). MM1S was a gift from Steven Rosen (Northwestern University or college). OPM1 and OPM2 were provided by Dr P. Leif Bersagel (Mayo Clinic-Tucson). S6B45 and KMS11 were kindly provided by Dr T. Kishimoto (Osaka University or college) and the Kawasaki Medical School, respectively. All MM cells were.
The CD138+ MM cell population probably also includes the side population, which is a CD138low+ subpopulation with stem cell properties
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