osteoprotegerin and Adenocarcinoma

Receptor activator of NF-κB (RANK), RANK ligand (RANKL) and osteoprotegerin (OPG) are key regulators of bone metabolism under both normal and pathological conditions, including prostate cancer (PCa) bone metastases. However, little is known concerning the expression and function of these regulators in prostate tumor samples and PCa cells and their correlation with invasion and bone metastasis. In the present study, we determined the expression of RANK, RANKL and OPG in 3 human PCa cell lines and 40 PCa patient samples by immunohistochemistry and enzyme-linked immunosorbent assay (ELISA). As controls, samples from 20 patients with benign prostate hyperplasia (BPH) and normal prostate epithelial RWPE2 cells were also included in the analyses. The effects of soluble RANKL (sRANKL) and OPG as well as RANK knockdown on PCa invasion were examined in Transwell assays. Immunohistochemical staining detected little RANK, OPG and RANKL expression in hyperplasia prostate while the percentages of positivity were increased to 50, 45 and 52.5%, respectively, in prostate tumor tissues. OPG and sRANKL levels in the prostate tumor samples as measured by ELISA were ~10-fold that in the BPHs (P<0.01) and the levels were higher in aggressive tumors than non-aggressive ones (P<0.05). The sRANKL level in the serum of PCa patients was the same as that in the patients with BPH, yet the serum OPG levels correlated with the tissue levels (R2=0.620, P<0.01, which both showed a 10-fold increase in PCa over BPH (P<0.01) with higher levels in aggressive PCa than non-aggressive ones (P<0.05). Consistent with the tissue analyses, expression levels of RANK mRNA and protein were detected in multiple human PCa cell lines by RT-PCR and western blotting, respectively. The treatment of PCa cells with RANKL significantly increased the number of invaded cells (P<0.01), which was suppressed by the decoy receptor OPG. RANK siRNA transfection dramatically dampened the stimulatory effect of RANKL on PCa cell invasion. Our findings indicate that the expression of RANK, RANKL and OPG may be used as diagnostic markers to identify patients at high risk for aggressive PCa and that the effective suppression of PCa cell migration by OPG via the blockage of RANKL activity represents a potential therapeutic strategy for interfering with prostate tumor metastasis and progression to bone.

Topics: Adenocarcinoma; Aged; Aged, 80 and over; Biomarkers, Tumor; Bone Neoplasms; Cell Line, Tumor; Humans; Male; Middle Aged; Neoplasm Invasiveness; Osteoprotegerin; Prostatic Neoplasms; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Risk

The tropism of breast cancer cells for bone and their tendency to induce an osteolytic phenotype are a result of interactions between breast cancer cells and stromal cells and are of paramount importance for bone metastasis. However, the underlying molecular mechanisms remain poorly understood. We hypothesize that tumor-stromal interaction alters gene expression in malignant tumor cells and stromal cells creating a unique expression signature that promotes osteolytic breast cancer bone metastasis and that inhibition of such interactions can be developed as targeted therapeutics. Microarray analysis was performed to investigate gene expression profiling at the tumor-bone (TB) interface versus the tumor alone area from syngenic mice injected with three different syngenic mammary tumor cell lines that differ in their metastatic potential. We identified matrix metalloproteinase 13 (MMP13), receptor activator of NF-kappaB ligand (RANKL), and integrins binding sialoprotein to be genes upregulated at the TB interface and validated. To determine the functional role of MMP13 in tumor-induced osteolysis, mice with Cl66 mammary tumors were treated with MMP13 antisense oligonucleotides (MMP13-ASO) or control scrambled oligonucleotides (control-ASO). Knockdown of MMP13 expression at the TB interface leads to significant reduction in bone destruction and in the number of activated osteoclasts at the TB interface. Further analysis to evaluate the mechanism of MMP13-dependent osteolytic bone metastasis revealed that MMP13-ASO treatment decreased active MMP9, RANKL levels, and transforming growth factor-beta signaling at the TB interface. Together, our data indicate that upregulation of MMP13 at the TB interface is important in tumor-induced osteolysis and suggest that MMP13 is a potential therapeutic target for breast cancer bone metastasis.

Topics: Adenocarcinoma; Animals; Bone and Bones; Bone Neoplasms; Cell Line, Tumor; Enzyme Activation; Female; Gene Expression Profiling; Mammary Neoplasms, Experimental; Matrix Metalloproteinase 13; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; Mice; Mice, Inbred BALB C; Osteoclasts; Osteolysis; Osteoprotegerin; RANK Ligand; RNA, Messenger; Signal Transduction; Transforming Growth Factor beta; Up-Regulation

Prostate cancers (PCas) produce factors that can serve as biomarkers for tumor metastasis and bone progression. Transduced GFP expression by cancer cells can be imaged to monitor therapy. We exploited both concepts by developing a GFP-expressing PCa cell line that expresses PTHrP and studying it in an animal model of malignancy with methods that assess the skeletal progression of this tumor.. We developed a GFP-producing PCa cell line by stable transduction of PC-3 PCa cells. This PC-3 variant was used to study tumor progression in an immunocompromised mouse model. Skeletal progression of the PCa cells and the effects of pamidronate administration were evaluated radiologically, fluorometrically, and by measurement of serum tumor markers.. The PC-3 cells produced extensive bone lesions when injected into the tibia of immunocompromised mice. The skeletal progression of the PC-3 cells could be monitored by GFP optical imaging, X-ray, and by measurements of tumor products in serum, notably PTHrP and OPG. Pamidronate treatment reduced tumor burden as assessed at autopsy by imaging and biomarkers.. Pamidronate treatment exhibited anti-tumor effects that were reflected by decreases in serum PTHrP, OPG, and by GFP and radiological imaging procedures. Imaging of GFP expression enables real-time monitoring of tumor growth in the bone. PTHrP and OPG may be useful as tumor biomarkers for PCa that has metastasized to bone. This novel human PCa model can be used to study the clinical potential of diagnostic and therapeutic modalities in the skeletal progression of PCas.

Topics: Adenocarcinoma; Animals; Antineoplastic Agents; Biomarkers, Tumor; Bone Neoplasms; Calcium; Carrier Proteins; Cell Line, Tumor; Diphosphonates; Disease Models, Animal; Glycoproteins; Green Fluorescent Proteins; Humans; Interleukin-6; Interleukin-8; Male; Membrane Glycoproteins; Mice; Mice, SCID; Microscopy, Fluorescence; Osteoprotegerin; Pamidronate; Parathyroid Hormone-Related Protein; Prostatic Neoplasms; Radiography; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Transduction, Genetic

Humoral hypercalcemia of malignancy (HHM) is mediated primarily by skeletal and renal responses to tumor-derived PTHrP. PTHrP mobilizes calcium from bone by inducing the expression of receptor activator for nuclear factor-kappaB ligand (RANKL), a protein that is essential for osteoclast formation, activation, and survival. RANKL does not influence renal calcium reabsorption, so RANKL inhibition is a rational approach to selectively block, and thereby reveal, the relative contribution of bone calcium to HHM. We used the RANKL inhibitor osteoprotegerin (OPG) to evaluate the role of osteoclast-mediated hypercalcemia in two murine models of HHM. Hypercalcemia was induced either by sc inoculation of syngeneic colon (C-26) adenocarcinoma cells or by sc injection of high-dose recombinant PTHrP (0.5 mg/kg, s.c., twice per day). In both models, OPG (0.2-5 mg/kg) caused rapid reversal of established hypercalcemia, and the speed and duration of hypercalcemia suppression were significantly greater with OPG (5 mg/kg) than with high-dose bisphosphonates (pamidronate or zoledronic acid, 5 mg/kg). OPG also caused greater reductions in osteoclast surface and biochemical markers of bone resorption compared with either bisphosphonate. In both models, hypercalcemia gradually returned despite clear evidence of ongoing suppression of bone resorption by OPG. These data demonstrate that osteoclasts and RANKL are important mediators of HHM, particularly in the early stages of the condition. Aggressive antiresorptive therapy with a RANKL inhibitor therefore might be a rational approach to controlling HHM.

Topics: Adenocarcinoma; Animals; Antineoplastic Agents; Bone Resorption; Calcium; Carrier Proteins; Cell Line, Tumor; Colonic Neoplasms; Diphosphonates; Disease Models, Animal; Glycoproteins; Humans; Hypercalcemia; Ligands; Membrane Glycoproteins; Mice; NF-kappa B; Osteoprotegerin; Pamidronate; Parathyroid Hormone-Related Protein; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor

Osteoprotegerin (OPG), identical with osteoclastogenesis inhibitory factor, is a member of a subgroup of the tumor necrosis factor (TNF)-receptor superfamily, which functions as a soluble decoy receptor. It has been reported that OPG expression is associated with bone metastasis of cancer of the breast and prostate. In the present study, we examined the expression of OPG in gastric carcinomas using immunohistochemistry and reverse-transcription polymerase chain reaction methods, and compared with clinicopathological parameters. The expression of OPG mRNA was confirmed in a gastric carcinoma cell line (MKN-7) and gastric carcinoma tissues. Immunohistochemically, strongly positive staining of OPG was found in 65% (67/103) of gastric carcinomas, whereas OPG protein was not detected in non-neoplastic mucosal epithelia. The expression of OPG protein in gastric carcinoma tissues correlates significantly with depth of tumor invasion, nodal metastases and advanced tumor stage. Furthermore, the prognosis of the cases with strong OPG expression was significantly worse than those with weak or no expression of OPG. These results suggest that OPG may participate in stomach carcinogenesis, invasion and metastasis, and may serve as a novel molecular marker for aggressive gastric cancer.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Female; Glycoproteins; Humans; Immunoenzyme Techniques; Male; Middle Aged; Osteoprotegerin; Prognosis; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Stomach Neoplasms; Survival Rate; Tumor Cells, Cultured

Receptor activator of NF-kappaB (RANK) activation induces several survival signals including nuclear factor kappaB (NF-kappaB) and activation and upregulation of Bcl-xL. The aim of this work was to determine whether RANK is expressed in oesophageal adenocarcinoma (EA) and its precursor, Barrett's metaplasia (BM). Sections of formalin-fixed and paraffin-embedded tissue from 23 oesophagectomy specimens showing EA and variable degrees of dysplasia in BM were immunohistochemically stained for RANK. All 23 carcinomas (100%) showed strong RANK immunoreactivity in most cancer cells. In 8 cases with high-grade dysplasia (HGD), RANK immunoreactivity was weaker and detected in a smaller percentage of cells. RANK was not detected in any area of BM that was negative for dysplasia (ND) (17 cases), or showed low-grade dysplasia (LGD) (9 cases). These results indicate that RANK is expressed as a late event during malignant progression in BM. Studies are needed to determine whether interfering with RANK signalling affects the survival of EA cells.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Barrett Esophagus; Disease Progression; Esophageal Neoplasms; Female; Glycoproteins; Humans; Immunohistochemistry; Male; Metaplasia; Middle Aged; Osteoprotegerin; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor

Certain malignancies, including breast cancer, frequently metastasize to bone, where the tumor cells induce osteoclasts to locally destroy bone. Osteoprotegerin (OPG), a member of the tumor necrosis factor receptor family, is a negative regulator of osteoclast differentiation, activation, and survival. We tested the ability of recombinant OPG to inhibit tumor-induced osteoclastogenesis, osteolysis, and skeletal tumor burden in two animal models. In a syngeneic model, mouse colon adenocarcinoma (Colon-26) cells were injected into the left ventricle of mice. Treatment with OPG dose-dependently decreased the number and area of radiographically evident lytic bone lesions, which, at the highest dose, were undetectable. Histologically, OPG also decreased skeletal tumor burden and tumor-associated osteoclasts. In a nude mouse model, OPG treatment completely prevented radiographic osteolytic lesions caused by human MDA-MB-231 breast cancer cells. Histologically, OPG decreased skeletal tumor burden by 75% and completely eradicated MDA tumor-associated osteoclasts. In both models, OPG had no effect on metastatic tumor burden in a panel of soft tissue organs. These data indicate that OPG may be an effective therapy for preventing osteolysis and decreasing skeletal tumor burden in patients with bone metastasis.

Topics: Adenocarcinoma; Animals; Bone Neoplasms; Breast Neoplasms; Cell Line, Transformed; Colonic Neoplasms; Disease Models, Animal; Dose-Response Relationship, Drug; Glycoproteins; Humans; Mice; Mice, Inbred BALB C; Mice, Inbred DBA; Mice, Nude; Osteolysis; Osteoprotegerin; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Xenograft Model Antitumor Assays