Receptor activator of nuclear factor-kappa B ligand (RANKL) directly modulates the gene expression profile of RANK-positive Saos-2 human osteosarcoma cells

Receptor activator of nuclear factor κB (RANK)/RANK ligand (RANKL)/osteoprotegerin (OPG) are the key regulators of bone metabolism. Recent findings demonstrated a crucial role of RANK in several bone-associated tumors. Indeed, we have recently demonstrated functional RANK expression both in a mouse and several human osteosarcoma cell lines. However, RANKL effects on osteosarcoma cells remain to be determined. In this study, we determined RANKL effects on RANK-positive Saos-2 human osteosarcoma cells. cDNA microarray and quantitative RT-PCR analyses clearly demonstrated that RANK-positive osteosarcoma cells were the target of RANKL as well as osteoclasts/osteoclast precursors. Thus, we present for the first time that RANKL can directly and significantly modulate gene expression of RANK-expressing Saos-2 cells. RANKL-modulated genes included genes that were implicated in protein metabolism, nucleic acid metabolism, intracellular transport, cytoskeleton organization and biogenesis, apoptosis and signaling cascade. Our results strengthen the involvement of the RANK/RANKL/OPG axis in osteosarcoma biology and capability to identify novel therapeutic approaches targeting RANK-positive osteosarcomas

Gene expression profiling in sinonasal adenocarcinoma

<p>Abstract</p> <p>Background</p> <p>Sinonasal adenocarcinomas are uncommon tumors which develop in the ethmoid sinus after exposure to wood dust. Although the etiology of these tumors is well defined, very little is known about their molecular basis and no diagnostic tool exists for their early detection in high-risk workers.</p> <p>Methods</p> <p>To identify genes involved in this disease, we performed gene expression profiling using cancer-dedicated microarrays, on nine matched samples of sinonasal adenocarcinomas and non-tumor sinusal tissue. Microarray results were validated by quantitative RT-PCR and immunohistochemistry on two additional sets of tumors.</p> <p>Results</p> <p>Among the genes with significant differential expression we selected <it>LGALS4, ACS5, CLU, SRI and CCT5 </it>for further exploration. The overexpression of <it>LGALS4, ACS5, SRI</it>, <it>CCT5 </it>and the downregulation of <it>CLU </it>were confirmed by quantitative RT-PCR. Immunohistochemistry was performed for LGALS4 (Galectin 4), ACS5 (Acyl-CoA synthetase) and CLU (Clusterin) proteins: LGALS4 was highly up-regulated, particularly in the most differentiated tumors, while CLU was lost in all tumors. The expression of ACS5, was more heterogeneous and no correlation was observed with the tumor type.</p> <p>Conclusion</p> <p>Within our microarray study in sinonasal adenocarcinoma we identified two proteins, LGALS4 and CLU, that were significantly differentially expressed in tumors compared to normal tissue. A further evaluation on a new set of tissues, including precancerous stages and low grade tumors, is necessary to evaluate the possibility of using them as diagnostic markers.</p

Towards zirconium phosphonate-based microarrays for probing DNA-protein interactions: critical influence of the location of the probe anchoring groups.

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Human osteosarcoma cells express functional receptor activator of nuclear factor-kappa B

RANK, RANK ligand (RANKL) and osteoprotegerin (OPG) are the key regulators of bone metabolism, both in normal and pathological conditions. Previous data have demonstrated that human osteosarcoma biopsies express RANKL as well as OPG, and functional RANK is expressed in a murine osteosarcoma cell line. As RANK expression in human osteosarcoma remains controversial, the aim of the present study was to analyse its expression in vitro in human osteosarcoma cell lines, ex vivo using pathological tissues, and then to determine its functionality in terms of signal transduction pathways modulated by RANKL. RT-PCR analysis and immunohistochemistry experiments revealed that RANK is expressed at both transcriptional and protein levels in MNNG/HOS, Saos-2 and MG-63 human osteosarcoma cell lines, in contrast to the U-2 OS osteosarcoma cell line and human osteoblasts, which were negative. RANK was also expressed in 57% of osteosarcoma biopsies. Furthermore, western blot experiments clearly demonstrated the functionality of RANK. Thus, RANKL significantly induced the phosphorylation of ERK1/2, p38 and IκB in RANK-positive osteosarcoma cells. This study is the first report of functional RANK expression in human osteosarcoma cells: this strengthens the involvement of the RANK–RANKL–OPG axis in primary bone tumour biology and identifies novel therapeutic approaches targeting RANK-positive osteosarcoma