Transcriptomic and proteomic analyses in bone tumor cells: Deciphering parathyroid hormone-related protein regulation of the cell cycle and apoptosis

Abstract

Giant cell tumor of bone (GCT) is an aggressive skeletal tumor characterized by local bone destruction, high recurrence rates, and metastatic potential. Previous works in our laboratory, including functional assays, have shown that neutralization of parathyroid hormone-related protein (PTHrP) in the cell environment inhibits cell proliferation and induces cell death in GCT stromal cells, indicating a role for PTHrP in cell propagation and survival. The objective of this study was to investigate the global gene and protein expression patterns of GCT cells in order to identify the underlying pathways and mechanisms of neoplastic proliferation provided by PTHrP in the bone microenvironment. Primary stromal cell cultures from 10 patients with GCT were used in this study. Cells were exposed to optimized concentrations of either PTHrP peptide or anti-PTHrP neutralizing antiserum and were analyzed with both cDNA microarray and proteomic microarray assays in triplicate. Hierarchical clustering and principal component analyses confirmed that counteraction of PTHrP in GCT stromal cells results in a clear-cut gene expression pattern distinct from all other treatment groups and the control cell line human fetal osteoblast (hFOB). Multiple bioinformatics tools were used to analyze changes in gene/protein expression and identify important gene ontologies and pathways common to this anti-PTHrP-induced regulatory gene network. PTHrP neutralization interferes with multiple cell survival and apoptosis signaling pathways by triggering both death receptors and cell cycle-mediated apoptosis, particularly via the caspase pathway, TRAIL pathway, JAK-STAT signaling pathway, and cyclin E/CDK2-associated G1/S cell cycle progression. These findings indicate that PTHrP neutralization exhibits anticancer potential by regulating cell-cycle progression and apoptosis in bone tumor cells, with the corollary being that PTHrP is a pro-neoplastic factor that can be targeted in the treatment of bone tumors. ß

1H NMR metabolomic signatures related to giant cell tumor of the bone

“…Interestingly, the metabolic pathway of alanine, aspartate, and glutamine also appears in the list of the differential pathways in previous transcriptomic analyses described for the GCTB; 48 in that study, the genes identied as aspartate oxidase, and glutamine-fructose-6-phosphate transaminase 2 by transcriptomics were identied as upregulated. 48 These observations together with our ndings strongly support a notion that the altered glutamine/glutamate ratio and aspartate metabolism leads to a disturbance in GCTB. In our study, the essential amino acids histidine and tryptophan are also upregulated in GCTB, supporting the elevation of amino acid synthesis in this tumor.…”

The Epigenetic Regulation of SOX9 by miR‐145 in Human Chondrosarcoma

“…Total RNA was isolated from the chondrosarcoma cell lines and control cell lines using the illustra RNAspin Mini RNA Isolation Kit (GE). Single‐stranded complementary DNA (cDNA) was synthesized from 1.0 µg of total RNA using the qScript cDNA SuperMix for RT–PCR (Quanta) as optimized in our lab [Mak et al, ]. Chondrosarcoma patient samples were harvested from human surgical specimens and homogenized in lysis buffer using a motorized rotor–stator homogenizer.…”

PTHLH (parathyroid hormone-like hormone)

“…In giant cell tumors, stromal cell production of PTHLH increased RANK ligand expression and led to giant cell formation (Cowan et al, 2012). PTHLH increased the survival of giant cell tumor stromal cells in vitro while the neutralization of PTHLH signaling induced cell death through the activation of different pathways (caspase, TRAIL, JAK-STAT, and cyclin E/CDK2) (Mak et al, 2012). PTHLH aids in normal mammary gland development and lactation as well as placental transfer of calcium.…”