epidermal-growth-factor and Bone-Neoplasms

Epidermal growth factor (EGF) signalling is well known for its multifaceted functions in development and tissue homoeostasis. The EGF family of ligands and receptors (ERBB family) have also been extensively investigated for their roles in promoting tumourigenesis and metastasis in a variety of cancer types. Recent findings indicate that EGF signalling is an important mediator of bone metastasis in breast, prostate and kidney cancers. The EGF signalling stimulates the growth of bone metastasis directly by increasing tumour cell proliferation and indirectly by engaging bone stromal cell in metastasis-promoting activities. Therefore, molecular targeting of ERBB receptors may benefit patients with bone metastasis and should be evaluated in clinical trials.

Topics: Animals; Bone Development; Bone Neoplasms; Epidermal Growth Factor; ErbB Receptors; Homeostasis; Humans; Osteolysis; Receptor, ErbB-2; Signal Transduction

Normal and abnormal developmental events in the prostate are strongly influenced by androgens. There is abundant evidence, however, that androgens are not the only substances present that have the capacity to influence prostatic growth. A number of polypeptides that either stimulate or inhibit growth have now been identified in the prostate. These include members of the HBGF family, TGF-beta family, EGF and TGF-alpha, PDGF, NGF, and the less well characterized osteoblast growth factors. In some cases, the prostatic cell population, stromal or epithelial, that synthesizes the growth factor and its receptor is known. This information and the properties of the growth factors suggest ways in which these polypeptides may be involved in regulating growth of the prostate, including benign prostatic hyperplasia and prostate cancer.

Topics: Bone Neoplasms; Cell Transformation, Neoplastic; Epidermal Growth Factor; Fibroblast Growth Factors; Growth Substances; Humans; Male; Neovascularization, Pathologic; Prostate; Receptors, Fibroblast Growth Factor; Transforming Growth Factor alpha; Transforming Growth Factor beta; Wound Healing

The value of tumor angiogenesis, EGFR and c-erbB-2 oncoprotein, a long with p 53 protein expression for predicting relapse-free survival was investigated in 110 node-negative breast cancer patients. The grade of neovascularization was assessed by the microvessel density which was obtained by an immunocytochemical staining by factor VIII-related antigen. EGFR, c-erbB-2 oncoprotein and p 53 oncoprotein were also determined by immunocytochemical assay. Univariate analysis showed no statistical significance of EGFR, c-erbB-2 and p53 status as a prognostic indicator. However, the microvessel density was a significant predictor of relapse-free survival. Patients with over 100 counts of factor VIII-RA positive cells per mm2 field in the most active areas of neovascularization showed significantly poorer prognosis compared to those with less than 100 counts (p < 0.005). Multivariate analysis demonstrated that microvessel density was an independent prognostic indicator in node-negative breast cancer patients (p < 0.0005). It was suggested that microvessel density might be of use in selecting the high-risk group in node-negative breast cancer patients needing adjuvant therapies.

Topics: Adenocarcinoma; Adenocarcinoma, Scirrhous; Axilla; Bone Neoplasms; Breast Neoplasms; Carcinoma, Papillary; Epidermal Growth Factor; Female; Humans; Immunohistochemistry; Lung Neoplasms; Lymph Nodes; Neovascularization, Pathologic; Prognosis; Receptor, ErbB-2; Regression Analysis; Tumor Suppressor Protein p53

Bone metastasis of colorectal cancer (CRC) cells leads to osteolysis. Aberrant activation of osteoclasts is responsible for bone resorption in tumor. In general, bone marrow-derived monocytes (BMMs) differentiate into osteoclasts, however, how CRC cells interact with BMMs and how to regulate the differentiation is elusive. We here report that CRC cells promote bone resorption in bone metastasis. Transcriptomic profiling revealed CCL3 up-regulated in MC-38 conditional medium treated BMMs. Further investigation demonstrated that CCL3 produced by BMMs facilitated cell infusion and thus promoted the osteoclastogenesis. In addition, CRC cells derived EGF stimulated the production of CCL3 in BMMs through activation of ERK/CREB pathway. Blockage of EGF or CCL3 can efficiently attenuate the osteolysis in bone metastasis of CRC.

Topics: Animals; Bone Neoplasms; Cell Communication; Cell Line, Tumor; Chemokine CCL3; Colorectal Neoplasms; Cyclic AMP Response Element-Binding Protein; Epidermal Growth Factor; Extracellular Signal-Regulated MAP Kinases; Male; Mice, Inbred C57BL; Osteoclasts; Osteogenesis; Osteolysis; Signal Transduction; Tibia

Ewing sarcoma (ES) is a type of childhood cancer probably arising from stem mesenchymal or neural crest cells. The epidermal growth factor receptor (EGFR) acts as a driver oncogene in many types of solid tumors. However, its involvement in ES remains poorly understood.. Human SK-ES-1 and RD-ES ES cells were treated with EGF, the EGFR inhibitor tyrphostin (AG1478), or phosphoinositide 3-kinase (PI3K) or extracellular-regulated kinase (ERK)/mitogen-activated kinase (MAPK) inhibitors. Cell proliferation survival, cycle, and senescence were analyzed. The protein content of possible targets of EGFR manipulation was measured by Western blot.. Cell proliferation and survival were increased by EGF and inhibited by AG1478. The EGFR inhibitor also altered the cell cycle, inducing arrest in G1 and increasing the sub-G1 population, reduced polyploidy and increased the population of senescent cells. In addition, AG1478 reduced the levels of phosphorylated AKT (p-AKT), ERK, p-ERK, cyclin D1, and brain-derived neurotrophic factor (BDNF), while enhancing p53 levels. Cell proliferation was also impaired by inhibitors of PI3K or ERK, alone or combined with AG1478.. Our findings reveal novel aspects of EGFR regulation of ES cells and provide early evidence for antitumor activities of EGFR inhibitors in ES.

Topics: Bone Neoplasms; Brain-Derived Neurotrophic Factor; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; G1 Phase Cell Cycle Checkpoints; Humans; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Quinazolines; Sarcoma, Ewing; Signal Transduction; Tumor Suppressor Protein p53; Tyrphostins

BACKGROUND The present work was performed to detect the potential inhibitory effect of cyclic guanosine monophosphate (cGMP)-dependent protein kinase II (PKG II) on epidermal growth factor (EGF) receptor-induced biological activity and related signal cascades in osteosarcoma cells. MATERIAL AND METHODS We transfected the osteosarcoma MG-63 cell line with an adenoviral vector encoding PKG II cDNA (Ad-PKGII) and incubated the transfected cells with 250 μM 8-pCPT-cGMP to activate the PKG II. We stimulated the MG-63 cells with100 ng/ml EGF, and then detected their proliferation using a CCK-8 assay. Transwell assay was used to examine MG-63 cell migration; and Western blot analysis was used to detect expression of matrix metalloproteinase 9 (MMP-9) and activation of ERK and Akt. RESULTS Stimulating cells by 100 ng/ml EGF promoted MG-63 cell proliferation and migration, ERK and Akt phosphorylation, and MMP-9 expression. These effects of EGF were inhibited in MG-63 cells infected with Ad-PKGII and incubated with 8-pCPT-cGMP. CONCLUSIONS Our results demonstrate that Ad-PKGII infection significantly inhibited EGF-induced proliferation and migration, as well as the associated-signal cascades; which indicates that PKG II might be a potential anti-cancer factor.

Topics: Bone Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclic GMP; Cyclic GMP-Dependent Protein Kinase Type II; Epidermal Growth Factor; ErbB Receptors; Humans; Osteosarcoma; Phosphorylation; Protein Binding; Signal Transduction; Transfection

BACKGROUND Epithelial-mesenchymal transition (EMT) is responsible for metastasis of cancers, and NF-κB can promote tumor progression. Ezrin is an important molecule participating in EMT. However, whether Ezrin mediates NF-κB in EGF-induced osteosarcoma is unknown. MATERIAL AND METHODS Ezrin phosphorylation, NF-κB activation, and EGF-induced EMT were studied in MG63 and U20S cells with NF-κB inhibition, silencing, or over-expressing Ezrin. Cell morphology, proliferation, migration, and motility were analyzed. An osteosarcoma model was established in mice by injecting MG63 and U20S and reducing Ezrin. RESULTS With EGF induction in vitro, Ezrin Tyr353 and Thr567 were phosphorylated, and EMT, proliferation, migration, and motility of osteosarcoma cells were promoted. Silencing Ezrin suppressed and over-expressing Ezrin promoted the nuclear translocation of p65 and phosphorylated IκBα (p-IκBα) in EGF-induced osteosarcoma cells. NF-κB inhibitor blocked EGF-induced EMT in both cell types, as well as reserving cell morphology and suppressing proliferation, migration, and motility. In vivo, reducing Ezrin significantly suppressed metastasis of osteosarcoma xenografts, increased liver and lung weights, and activated NF-κB, which were both induced by EGF. CONCLUSIONS Ezrin/NF-κB regulated EGF-induced EMT, as well as progression and metastasis of osteosarcoma in vivo and in vitro. Ezrin/NF-κB may be a new therapeutic target to prevent osteosarcoma from deterioration.

Topics: Animals; Bone Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cytoskeletal Proteins; Disease Models, Animal; Disease Progression; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; Heterografts; Male; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; NF-kappa B; NF-KappaB Inhibitor alpha; Osteosarcoma; Phosphorylation; Signal Transduction

Osteosarcoma, the most common primary bone tumor, occurs most frequently in children and adolescents and has a 5-year survival rate, which is unsatisfactory. As epidermal growth factor receptor (EGFR) positively correlates with TNM (tumor-node-metastasis) stage in osteosarcoma, EGFR may play an important role in its progression. The purpose of this study was to explore potential mechanisms underlying this correlation. We found that EGF promotes MG63 cell migration and invasion as well as stress fiber formation via Rho A activation and that these effects can be reversed by inhibiting Rho A expression. In addition, molecules downstream of Rho A, including ROCK1, LIMK2, and Cofilin, are activated by EGF in MG63 cells, leading to actin stress fiber formation and cell migration. Moreover, inhibition of ROCK1, LIMK2, or Cofilin in MG63 cells using known inhibitors or short hairpin RNA (shRNA) prevents actin stress fiber formation and cell migration. Thus, we conclude that Rho A/ROCK1/LIMK2/Cofilin signaling mediates actin microfilament formation in MG63 cells upon EGFR activation. This novel pathway provides a promising target for preventing osteosarcoma progression and for treating this cancer.

Topics: Bone Neoplasms; Cell Line, Tumor; Cell Movement; Cofilin 1; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Neoplastic; Humans; Lim Kinases; Osteosarcoma; rho-Associated Kinases; rhoA GTP-Binding Protein; Signal Transduction; Stress Fibers

Approximately 70% of prostate cancer patients will develop bone metastasis in axial and other regions of the skeleton. Epidermal growth factor (EGF) generated from bone tissue contributes to prostate cancer metastasis. In a previous study, penta-O-galloyl-β-D-glucose (PGG) suppressed androgen-independent prostate cancer bone metastasis by transcriptionally repressing EGF-induced MMP-9 expression. This study utilized proteomics to analyze the effects of PGG in EGF-induced prostate cancer bone metastasis. This study showed that PGG suppressed EGF-induced eIF3i expression in PC-3 cells. By transfection of eIF3i shRNA, it was observed that reduced eIF3i expression suppressed the invasion of PC-3 cells in vitro. PGG reduced EGF-induced eIF3i expression through inhibition of the PI3K/AKT/mTOR pathway. Therefore, PGG may be able to be used as a potential new therapeutic drug for prostate cancer bone metastasis.

Topics: Bone Neoplasms; Cell Line, Tumor; Epidermal Growth Factor; Eukaryotic Initiation Factor-3; Gene Expression; Humans; Hydrolyzable Tannins; Male; Neoplasm Metastasis; Phosphoinositide-3 Kinase Inhibitors; Prostatic Neoplasms; Proto-Oncogene Proteins c-akt; RNA, Small Interfering; TOR Serine-Threonine Kinases; Transfection

The molecular regulation of the invasion of osteosarcoma (OS) remains elusive. Here, we reported significant lower level of miR-143 and significant levels of phosphorylated EGFR and MMP9 in the resected OS from the patients, compared to the adjacent normal tissue. Moreover, strong correlation was detected among these three factors. We thus hypothesized existence of a causal link, which prompted us to use two human OS cell lines to study the interaction of miR-143, MMP9, and activation of EGFR signaling. We found that EGF-induced EGFR phosphorylation in both lines activated MMP9, and consequently cancer invasiveness. Both an inhibitor for EGFR phosphorylation and an inhibitor for ERK1/2 phosphorylation significantly inhibited the EGF-induced activation of MMP9. Moreover, miR-143 levels did not alter by EGF-induced EGFR phosphorylation, while overexpression of miR-143 antagonized EGF-induced MMP9 activation without affecting EGFR phosphorylation. Taken together, our data suggest that miR-143 inhibits EGFR signaling through its downstream ERK/MAPK signaling cascades to control MMP9 expression in OS. Thus, miR-143, EGFR, and MMP9 are therapeutic targets for inhibiting OS invasion.

Topics: Bone Neoplasms; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Gene Expression; Gene Expression Regulation, Neoplastic; Humans; MAP Kinase Signaling System; Matrix Metalloproteinase 9; MicroRNAs; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neoplasm Invasiveness; Osteosarcoma; Phosphorylation; Signal Transduction

To explore the effects of a knockdown of cyclooxygenase 2 upon the growth and migration of SaOS2 cells.. We employed lentivirus mediated-RNA interference technology to knockdown the endogenous expression of gene COX-2 in human osteosarcoma cells (SaOS2) and analyzed their phenotypical changes. The effects of COX-2 silencing on the proliferation, cell cycle and migration of SaOS2 cells were assessed by thiazolyl blue tetrazolium bromide, flow cytometry and migration assays respectively. COX-2, vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) mRNA and protein expression were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot.. A decreased expression of COX-2 in human osteosarcoma cells significantly inhibited the growth and decreased the migration ability of SaOS2 cells. Furthermore, it also reduced VEGF, EGF and bFGF mRNA and protein expression.. The COX-2 signaling pathway may provide a novel therapeutic target for the treatment of human osteosarcoma.

Topics: Bone Neoplasms; Cell Line, Tumor; Cyclooxygenase 2; Epidermal Growth Factor; Fibroblast Growth Factor 2; Gene Knockdown Techniques; Humans; Osteosarcoma; RNA Interference; RNA, Small Interfering; Signal Transduction; Vascular Endothelial Growth Factor A

Osteosarcoma (OS) is a kind of malignancy wherein the tumor cells form malignant bone-like or bone tissue. Tenascin-C (TN-C), an important extracellular matrix (ECM) protein, plays an indispensable role in tumor development. However, its regulatory factors, expression, and function in OS pathological process have not been studied extensively. Expression of TN-C is induced by growth factors as well as mechanical strain in fibroblast. So we asked whether mechanical stain and growth factors could induce TN-C expression in OS as well as which pathways were involved in those processes. We found that when mechanical strain was applied to OS cells cultured on silicone membrane, TN-C mRNA and protein levels were increased 10-fold within 8 h compared to the resting control. Likewise, when epidermal growth factors (EGFs) and insulin-like growth factor (IGF-1) were added to cells, TN-C mRNA levels increased six fold and eightfold, respectively, within 24h compared to the control. Growth factors (EGF and IGF-1) and mechanical strain had additive effects on the induction of TN-C mRNA expression in OS. Both ROCK-I/II inhibitor and MEK-1 inhibitor inhibited TN-C induction by EGF or IGF-1, while only ROCK-I/II inhibitor had a strong subdued effect on TN-C induction by mechanical strain. Taken together, our findings suggest that growth factors and mechanical strain can induce TN-C in OS through different pathways additively.

Topics: Bone Neoplasms; Cell Line, Tumor; Epidermal Growth Factor; Fibroblasts; Gene Expression Regulation, Neoplastic; Humans; MAP Kinase Kinase 1; Mechanotransduction, Cellular; Osteosarcoma; Protein Kinase Inhibitors; rho-Associated Kinases; Somatomedins; Stress, Physiological; Tenascin; Up-Regulation

Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) show dramatic antitumor activity in a subset of patients with non-small cell lung cancer who have an active mutation in the epidermal growth factor receptor (EGFR) gene. On the other hand, some lung cancer patients with wild type EGFR also respond to EGFR-TKIs, suggesting that EGFR-TKIs have an effect on host cells as well as tumor cells. However, the effect of EGFR-TKIs on host microenvironments is largely unknown. A multiple organ metastasis model was previously established in natural killer cell-depleted severe combined immunodeficient mice using human lung cancer cells. This model was used to investigate the therapeutic efficacy of erlotinib, an EGFR-TKI, on multiple organ metastases induced by human small cell lung cancer cells (SBC-5 cells) that did not express EGFR. Although erlotinib did not have any effect on the proliferation of SBC-5 cells in vitro, it significantly suppressed bone and lung metastases in vivo, but not liver metastases. An immunohistochemical analysis revealed that, erlotinib significantly suppressed the number of osteoclasts in bone metastases, whereas no difference was seen in microvessel density. Moreover, erlotinib inhibited EGF-induced receptor activator of nuclear factor kappa-B expression in an osteoblastic cell line (MC3T3-E1 cells). These results strongly suggested that erlotinib prevented bone metastases by affecting host microenvironments irrespective of its direct effect on tumor cells.

Topics: Animals; Bone Neoplasms; Carcinoma, Small Cell; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epidermal Growth Factor; ErbB Receptors; Erlotinib Hydrochloride; Humans; Lung Neoplasms; Male; Mice; Neoplasm Metastasis; Neovascularization, Pathologic; Osteoblasts; Osteoclasts; Protein Kinase Inhibitors; Quinazolines; RANK Ligand

HER2 signalling by heterodimerisation with EGFR and HER3 in breast cancer is associated with worst outcome of the afflicted patients, which is attributed not only to the aggressiveness of such tumours but also to therapy resistance. Thus, in the present study we investigated the role of EGFR, HER2 and HER3 lateral signalling in cell migration by applying the MDA-MB-468-HER2 (MDA-HER2) breast cancer cell line, representing a valid model system. Knockdown of HER3 expression by siRNA resulted in decreased phosphorylated AKT (pAKT) levels, abrogated epidermal growth factor (EGF)-mediated PLC-γ1 activation and a diminished EGF-induced migratory activity, depicting the interplay of EGF receptor (EGFR)/HER2/PLC-γ1 and HER2/HER3/PI3K signalling in mediating the migration of EGFR/HER2/HER3-expressing breast cancer cells. Since therapy failure usually arises from metastatic cells, we further investigated whether HER3 signalling was active in established breast cancer disseminated tumour cell (DTC) lines as well as in primary DTCs derived from breast cancer patients. EGF treatment of DTC lines resulted solely in increased pAKT S473 levels, whereas in MDA-HER2 cells both pAKT S473 and pAKT T308 levels were increased upon EGF stimulation. Moreover, despite active HER3 molecules, as indicated by pTyr1222 staining, about 90% of analysed breast cancer patient DTCs exhibited very low or even no detectable pAKT S473 levels, suggesting that these cells might have fallen into dormancy. In summary, our data indicate the important role in EGFR, HER2 and HER3 lateral signalling in breast cancer cell migration. Moreover, our data further show that primary tumour cells and DTCs can vary in their HER activation status, which is important to know in the context of cancer therapy.

Topics: Animals; Bone Neoplasms; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Mice; Mice, Inbred BALB C; Neoplasm Invasiveness; Phosphatidylinositol 3-Kinase; Phospholipase C gamma; Phosphorylation; Proto-Oncogene Proteins c-akt; Receptor, ErbB-2; Receptor, ErbB-3; RNA Interference; Signal Transduction; Transfection

Functional interactions between cancer cells and the bone microenvironment contribute to the development of bone metastasis. Although the bone metastasis of prostate cancer is characterized by increased ossification, the molecular mechanisms involved in this process are not fully understood. Here, the roles of bone morphogenetic proteins (BMPs) in the interactions between prostate cancer cells and bone stromal cells were investigated. In human prostate cancer LNCaP cells, BMP-4 induced the production of Sonic hedgehog (SHH) through a Smad-dependent pathway. In mouse stromal MC3T3-E1 cells, SHH up-regulated the expression of activin receptor IIB (ActR-IIB) and Smad1, which in turn enhanced BMP-responsive reporter activities in these cells. The combined stimulation with BMP-4 and SHH of MC3T3-E1 cells cooperatively induced the expression of osteoblastic markers, including alkaline phosphatase, bone sialoprotein, collagen type II α1, and osteocalcin. When MC3T3-E1 cells and LNCaP cells were co-cultured, the osteoblastic differentiation of MC3T3-E1 cells, which was induced by BMP-4, was accelerated by SHH from LNCaP cells. Furthermore, LNCaP cells and BMP-4 cooperatively induced the production of growth factors, including fibroblast growth factor (FGF)-2 and epidermal growth factor (EGF) in MC3T3-E1 cells, and these may promote the proliferation of LNCaP cells. Taken together, our findings suggest that BMPs provide favorable circumstances for the survival of prostate cancer cells and the differentiation of bone stromal cells in the bone microenvironment, possibly leading to the osteoblastic metastasis of prostate cancer.

Topics: Activin Receptors, Type II; Animals; Antigens, Differentiation; Bone Morphogenetic Protein 4; Bone Neoplasms; Calcinosis; Cell Line, Tumor; Epidermal Growth Factor; Fibroblast Growth Factor 2; Humans; Male; Mice; Neoplasm Proteins; Osteoblasts; Prostatic Neoplasms; Signal Transduction; Smad1 Protein; Stromal Cells; Tumor Microenvironment

Versican is detected in the interstitial tissues at the invasive margins of breast carcinoma, is predictive of relapse, and negatively impacts overall survival rates. The versican G3 domain is important in breast cancer cell growth, migration and bone metastasis. However, mechanistic studies evaluating versican G3 enhanced breast cancer bone metastasis are limited.. A versican G3 construct was exogenously expressed in the 66c14 and the MC3T3-E1 cell line. Cells were observed through light microscopy and viability analyzed by Coulter Counter or determined with colorimetric proliferation assays. The Annexin V-FITC apoptosis detection kit was used to detect apoptotic activity. Modified Chemotactic Boyden chamber migration invasion assays were applied to observe tumor migration and invasion to bone stromal cells and MC3T3-E1 cells. Alkaline phosphatase (ALP) staining and ALP ELISA assays were performed to observe ALP activity in MC3T3-E1 cells.. In the four mouse breast cancer cell lines 67NR, 66c14, 4T07, and 4T1, 4T1 cells expressed higher levels of versican, and showed higher migration and invasion ability to MC3T3-E1 cells and primary bone stromal cells. 4T1 conditioned medium (CM) inhibited MC3T3-E1 cell growth, and even lead to apoptosis. Only 4T1 CM prevented MC3T3-E1 cell differentiation, noted by inhibition of alkaline phosphatase (ALP) activity. We exogenously expressed a versican G3 construct in a cell line that expresses low versican levels (66c14), and observed that the G3-expressing 66c14 cells showed enhanced cell migration and invasion to bone stromal and MC3T3-E1 cells. This observation was prevented by selective EGFR inhibitor AG1478, selective MEK inhibitor PD 98059, and selective AKT inhibitor Triciribine, but not by selective JNK inhibitor SP 600125. Versican G3 enhanced breast cancer cell invasion to bone stromal cells or osteoblast cells appears to occur through enhancing EGFR/ERK or AKT signaling. G3 expressing MC3T3-E1 cells showed inhibited cell growth and cell differentiation when cultured with TGF-β1 (1 ng/ml), and expressed enhanced cell apoptosis when cultured with TNF-α (2 ng/ml). Enhanced EGFR/JNK signaling appears to be responsible for G3 enhanced osteoblast apoptosis and inhibited osteoblast differentiation. Whereas repressed expression of GSK-3β (S9P) contributes to G3 inhibited osteoblast growth. Versican G3 functionality was dependent on its EGF-like motifs. Without the structure of EGF-like repeats, the G3 domain would not confer enhancement of tumor cell migration and invasion to bone with concordant inhibition of osteoblast differentiation and promotion of osteoblast apoptosis.. Versican enhances breast cancer bone metastasis not only through enhancing tumor cell mobility, invasion, and survival in bone tissues, but also by inhibiting pre-osteoblast cell growth, differentiation, which supply favorable microenvironments for tumor metastasis.

Topics: Amino Acid Motifs; Animals; Apoptosis; Bone Neoplasms; Breast Neoplasms; Cell Differentiation; Cell Line, Tumor; Cell Movement; Cell Proliferation; Culture Media, Conditioned; Epidermal Growth Factor; Female; Gene Expression; Humans; Mice; Models, Biological; Neoplasm Metastasis; Osteoblasts; Protein Interaction Domains and Motifs; Versicans

Bone metastasis occurs frequently in advanced prostate cancer (PCa) patients; however, it is not known why this happens. The epidermal growth factor receptor (EGFR) ligand EGF is available to early stage PCa; whereas, TGF-alpha is available when PCa metastasizes. Since the microenvironment of metastases has been shown to play a role in the survival of the tumor, we examined whether the ligands had effects on cell survival and proliferation in early and late PCa.. We used LNCaP cells as a model of early stage, non-metastatic PCa and the isogenic C4-2B cells as a model of late stage, metastatic PCa.. We found that the proliferation factor MAPK and the survival factor AKT were differentially activated in the presence of different ligands. TGF-alpha induced growth of C4-2B cells and not of the parental LNCaP cells; however, LNCaP cells expressing a constitutively active AKT did proliferate with TGF-alpha. Therefore, AKT appeared to be the TGF-alpha-responsive factor for survival of the late stage PCa cells. LNCaP cells exposed to EGF produced more osteoprotegerin (OPG), an inhibitor of bone remodeling, than C4-2B cells with TGF-alpha, which had increased expression of RANKL, an activator of bone remodeling. In concordance, TGF-alpha-treated C4-2B conditioned medium was able to differentiate an osteoclast precursor line to a greater extent than EGF-treated C4-2B or TGF-alpha-treated LNCaP conditioned media.. The switch in EGFR ligand availability as PCa progresses affects cell survival and contributes to bone remodeling.

Topics: Adenocarcinoma; Bone Neoplasms; Bone Remodeling; Cell Line, Tumor; Disease Progression; Epidermal Growth Factor; ErbB Receptors; Humans; Ligands; Male; Mitogen-Activated Protein Kinase Kinases; Models, Biological; Neoplasm Staging; Prostatic Neoplasms; Proto-Oncogene Proteins c-akt; RANK Ligand; Signal Transduction; Transforming Growth Factor alpha

Prostate carcinoma is the most frequently diagnosed malignancy and the second leading cause of cancer-related death of men in the United States. Epidermal growth factor (EGF) generated from bone tissue contributes to prostate cancer metastasis through stimulating matrix metalloproteinase (MMP) secretions from prostate cancer cells. In this study, in vitro invasion assay was performed by incubating penta-O-galloyl-beta-D-glucose (5GG) at various concentrations with 2 x 10(4) PC-3 cells for 48 h. The anti-invasive and cytotoxic effects of 5GG were found and evaluated on the human androgen-independent prostate cancer PC-3 cell line by MTT assays and Western blot analyses. 5GG inhibited the EGF-induced cell invasiveness and MMP-9 expression in a dose- and time-dependent manner by reducing the MMP-9 transcriptional activity. To explore the mechanisms for the 5GG-mediated regulation of MMP-9, we further examined the effects of 5GG on transcription factors, including NF-kappaB, AP-1, and mitogen-activated protein kinase (MAPK) activities. The results showed that 5GG suppressed the EGF-induced NF-kappaB nuclear translocation and also abrogated the EGF-induced activation of c-jun N-terminal kinase (JNK), an upstream modulator of NF-kappaB. Moreover, we showed that 5GG reduced EGFR expression through the proteasome pathway. These results suggest that 5GG may exert at least part of its anti-invasive effect in androgen-independent prostate cancer by controlling MMP-9 expression through the suppression of the EGFR/JNK pathway. Finally, 5GG suppresses invasion and tumorigenesis in nude mice treatment with intratibia injection of PC-3 cells. These in vitro and in vivo results suggest that 5GG may be a therapeutic candidate for the treatment of advanced prostate cancer.

Topics: Animals; Bone Neoplasms; Cell Line, Tumor; Down-Regulation; Epidermal Growth Factor; ErbB Receptors; Humans; Hydrolyzable Tannins; Male; Matrix Metalloproteinase 9; Mice; Mice, Nude; Neoplasm Invasiveness; Neoplasm Transplantation; Prostatic Neoplasms; RNA, Messenger; Transcription, Genetic

Bone metastasis is mediated by complex interactions between tumor cells and resident stromal cells in the bone microenvironment. The functions of metalloproteinases in organ-specific metastasis remain poorly defined despite their well-appreciated role in matrix degradation and tumor invasion. Here, we show a mechanism whereby two distinct metalloproteinases, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS1) and matrix metalloproteinase-1 (MMP1), orchestrate a paracrine signaling cascade to modulate the bone microenvironment in favor of osteoclastogenesis and bone metastasis. Proteolytic release of membrane-bound epidermal growth factor (EGF)-like growth factors, including Amphiregulin (AREG), heparin-binding EGF (HB-EGF), and transforming growth factor alpha (TGFalpha) from tumor cells suppress the expression of osteoprotegerin (OPG) in osteoblasts and subsequently potentiate osteoclast differentiation. EGF receptor (EGFR) inhibitors block osteolytic bone metastasis by targeting EGFR signaling in bone stromal cells. Furthermore, elevated MMP1 and ADAMTS1 expression is associated with increased risk of bone metastasis in breast cancer patients. This study established MMP1 and ADAMTS1 in tumor cells, as well as EGFR signaling in osteoblasts, as promising therapeutic targets for inhibiting bone metastasis of breast cancer.

Topics: ADAM Proteins; ADAMTS1 Protein; Animals; Bone and Bones; Bone Neoplasms; Breast Neoplasms; Cell Differentiation; Cell Line; Cell Proliferation; Epidermal Growth Factor; Female; Gefitinib; Gene Expression; Gene Expression Regulation, Neoplastic; Gene Silencing; Matrix Metalloproteinase 1; Mice; Mice, Nude; Osteoblasts; Osteoprotegerin; Protein Kinase Inhibitors; Quinazolines; RANK Ligand; Signal Transduction

Modulation of apoptosis may influence sarcoma pathogenesis and/or aggressiveness. The Fas death pathway, mediated by FasL or TGFbeta, is one of two apoptotic pathways. Recent studies report that EGF can modulate TGFbeta and/or FasL expression/activity; thus, EGF has the potential to influence activation of the Fas pathway. EGF is not always detectable in mesenchymal tumors; therefore, we hypothesized EGF would define which Fas ligand predominates. We assayed 57 surgically removed human sarcomas for 10 genes involved in the Fas pathway. Skeletal muscle biopsies from eight patients served as controls. Sample transcripts were detected by real-time RT-PCR. We attempted to identify relevant predictor variables. The 57 sarcomas were segregated into two categories defined by EGF mRNA content: (1) 23 tumors with EGF concentrations that approximated muscle EGF transcript levels (high-EGF tumors); and (2) 34 tumors that either lacked EGF mRNA, or whose mRNA levels were very low and frequently undetected by PCR (low-EGF tumors). TGFbeta1 expression best predicted Fas transcript concentrations in the 34 low-EGF sarcomas, while FasL predicted Fas mRNA levels in the remaining 23 high-EGF sarcomas. The results suggest ligand activity in the Fas death pathway correlates with EGF transcription in sarcomas.

Topics: Apoptosis; Bone Neoplasms; Caspase 10; Caspase 8; Death Domain Receptor Signaling Adaptor Proteins; Epidermal Growth Factor; Fas Ligand Protein; Fas-Associated Death Domain Protein; Humans; Muscle, Skeletal; Regression Analysis; RNA, Messenger; Sarcoma; Signal Transduction; Transcription, Genetic; Transforming Growth Factor beta

To identify regulators of the cartilaginous phenotype, on the basis of their differential expression in human conventional chondrogenic tumors compared with articular cartilage.. Differential proteomics analysis revealed matrilin-3 (MATN3) as a candidate regulator of the cartilaginous phenotype. Its capacity to modulate gene expression was investigated in human HCS-2/8 chondrosarcoma cells and transfected chondrocytes, using cell culture fractionation, reverse transcription-polymerase chain reaction, and Western blot analyses.. Increased expression of the cartilage-specific matrix protein MATN3 was specifically observed in enchondromas and conventional chondrosarcomas. A substantial fraction of MATN3 was found in cytoplasmic structures of tumor cells, as demonstrated by immunohistochemistry. Analyses of intracellular MATN3 revealed that it corresponded to an imperfectly maturated MATN3 polypeptide, both in HCS-2/8 human chondrosarcoma cells and in transfected human chondrocytes. Moderately increased expression of MATN3 resulted in its intracellular retention. Antibody-mediated blockade of soluble, extracellular MATN3 in HCS-2/8 cell cultures resulted in increased expression of MATN3 and the chondrogenic transcription factor SOX9. Conversely, increased ectopic expression of MATN3 resulted in decreased expression of MATN3 and SOX9 in primary chondrocytes, while a mutant MATN3 lacking its first epidermal growth factor (EGF)-like domain failed to down-regulate SOX9.. Aberrant expression and processing of MATN3 are hallmarks of conventional cartilaginous neoplasms. A particular step in the maturation of MATN3 limits its processing through the secretion machinery, resulting in its intracellular accumulation upon increased expression. Soluble, secreted MATN3, however, down-regulates SOX9 at the messenger RNA and protein levels. The first EGF-like domain of MATN3 is a critical determinant of its regulatory activity toward SOX9. These activities of MATN3 suggest that its increased expression in osteoarthritis might contribute to the degeneration of articular cartilage.

Topics: Adolescent; Adult; Aged; Blotting, Western; Bone Neoplasms; Cartilage, Articular; Cell Differentiation; Cells, Cultured; Child; Child, Preschool; Chondrocytes; Chondroma; Chondrosarcoma; Down-Regulation; Epidermal Growth Factor; Extracellular Matrix Proteins; Female; Flow Cytometry; Humans; Immunohistochemistry; Male; Matrilin Proteins; Middle Aged; Osteoarthritis; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; SOX9 Transcription Factor

To investigate, using prostate needle-biopsy specimens at diagnosis from patients with bone metastatic prostate cancer, whether the relationship between neuroendocrine (NE) cell differentiation and human epidermal growth factor-2 (HER-2) expression is a prognostic factor for outcome.. The study included 50 patients diagnosed as having bone metastatic prostate cancer between January 1998 and December 2001. We tested for NE cell differentiation by using immunohistochemical (IHC) staining for chromogranin A (CgA), and for HER-2, using a commercial test for IHC staining.. Eleven patients (22%) were positive for CgA; there was a significant difference in the time to recurrence (P = 0.025) but no significant differences in cause-specific survival rate or survival rate after recurrence. In all, 21 patients (42%) were positive for HER-2; the cause-specific survival rate, time to recurrence and survival rate after recurrence were all significantly more favourable in the HER-2-negative group (P = 0.008, 0.049 and 0.025, respectively). In the 49 patients for whom both factors could be determined, there was no significant correlation between CgA and HER-2 positivity.. NE cell differentiation of the primary tumour in patients with bone metastatic prostate cancer does not reflect the prognosis, whereas HER-2 overexpression is a prognostic factor for an unfavourable outcome. These results suggest that NE cell differentiation is not induced by HER-2.

Topics: Aged; Aged, 80 and over; Biopsy, Needle; Bone Neoplasms; Chromogranin A; Epidermal Growth Factor; Humans; Immunohistochemistry; Male; Middle Aged; Prognosis; Prostate-Specific Antigen; Prostatic Neoplasms; Receptor, ErbB-2

Epidermal growth factor (EGF)-like ligands and their receptors constitute one of the most important signaling networks functioning in normal tissue development and cancer biology. Recent in vivo mouse models suggest this signaling network plays an essential role in bone metabolism. Using a coculture system containing bone marrow macrophage and osteoblastic cells, here we report that EGF-like ligands stimulate osteoclastogenesis by acting on osteoblastic cells. This stimulation is not a direct effect because osteoclasts do not express functional EGF receptors (EGFRs). Further studies reveal that EGF-like ligands strongly regulate the expression of two secreted osteoclast regulatory factors in osteoblasts by decreasing osteoprotegerin (OPG) expression and increasing monocyte chemoattractant protein 1 (MCP1) expression in an EGFR-dependent manner and consequently stimulate TRAP-positive osteoclast formation. Addition of exogenous OPG completely inhibited osteoclast formation stimulated by EGF-like ligands, while addition of a neutralizing antibody against MCP-1 exhibited partial inhibition. Coculture with bone metastatic breast cancer MDA-MB-231 cells had similar effects on the expression of OPG and MCP1 in the osteoblastic cells, and those effects could be partially abolished by the EGFR inhibitor PD153035. Because a high percentage of human carcinomas express EGF-like ligands, our findings suggest a novel mechanism for osteolytic lesions caused by cancer cells metastasizing to bone.

Topics: Animals; Bone Neoplasms; Cells, Cultured; Chemokine CCL2; Coculture Techniques; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation; Humans; Ligands; Mice; Osteoblasts; Osteoclasts; Osteolysis; Osteoprotegerin; RANK Ligand; Signal Transduction

Susceptibility to extracellular matrix and growth factors has been demonstrated to play a critical role in the development of prostate cancer (PCa) metastases. The aim of this study was to elucidate some mechanisms by which stroma controls tumor progression.. In our study we tested the growth ability of the LNCaP human prostatic cell line in steroid-free culture conditions in response to osteopontin (OPN), a non-collageneous matrix protein, localized in large amounts in the bone.. In the LNCaP cell model, OPN stimulates cell proliferation in serum-free medium and colony growth at high dilution but this effect is visible only in presence of epidermal growth factor (EGF). Proliferation induced by OPN is accompanied by a sustained activation of EGF receptor (EGFR) whose phosphorylation is detectable up to 12 hr after treatment in association with EGF. The colocalization of integrin beta1, a ligand of OPN, and of EGFR on the cellular membrane, suggests that the association of these cell surface receptors may be the principal mechanism involved in the long-term activation of the EGFR.. Our data describe a new possible mechanism involved in the establishment of bone metastases which may also account for the formation of androgen-independent cellular clones, frequently responsible of the clinical progression of PCa.

Topics: Bone Neoplasms; Cell Adhesion; Cell Division; Cytokines; Disease Progression; Epidermal Growth Factor; ErbB Receptors; Humans; Male; Osteopontin; Phosphoproteins; Prostatic Neoplasms; Sialoglycoproteins; Stromal Cells

Renal cell carcinoma (RCC) frequently produces metastases to the musculoskeletal system that are a major source of morbidity in the form of pain, immobilization, fractures, neurological compromise, and a decreased ability to perform activities of daily living. Patients with metastatic RCC therefore have a dismal prognosis because there is no effective adjuvant treatment for this disease. Because the epidermal growth factor receptor (EGF-R) signaling cascade is important in the growth and metastasis of RCC, its blockade has been hypothesized to inhibit tumor growth and hence prevent resultant bone destruction. We determined whether blockade of EGF-R by the tyrosine kinase inhibitor PKI 166 inhibited the growth of RCC in bone. We use a novel cell line, RBM1-IT4, established from a human RCC bone metastasis. Protein and mRNA expression of the ligands and receptors was assessed by Western and Northern blots. The stimulation of RBM1-IT4 cells with epidermal growth factor or transforming growth factor alpha resulted in increased cellular proliferation and tyrosine kinase autophosphorylation. PKI 166 prevented these effects. First, RBM1-IT4 cells were implanted into the tibia of nude mice, where they established lytic, progressively growing lesions, after which the mice were treated with PKI 166 alone or in combination with paclitaxel (Taxol). Immunohistochemical analysis revealed that tumor cells and tumor-associated endothelial cells in control mice expressed activated EGF-R. Treatment of mice with PKI 166 alone or in combination with Taxol produced a significant decrease in the incidence and size of bone lesions as compared with the results in control or Taxol-treated mice (P < 0.001). Treatment with PKI 166 also decreased the expression of phosphorylated EGF-R by tumor cells and tumor-associated endothelial cells, and this was even more pronounced with PKI 166 plus Taxol treatment. The PKI 166 plus Taxol combination produced apoptosis of tumor cells and tumor-associated endothelial cells. Tumor cell proliferation, shown by proliferating cell nuclear antigen positivity, was decreased in all treatment groups. In addition, the integrity of the bone was maintained in mice treated with PKI 166 or PKI 166 plus Taxol, whereas massive bone destruction was seen in control and Taxol-treated mice. These results suggest that blockade of EGF-R signaling inhibits growth of RCC in the bone by its effect on tumor cells and tumor-associated endothelial cells.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Bone Neoplasms; Carcinoma, Renal Cell; Cell Division; Epidermal Growth Factor; ErbB Receptors; Humans; Kidney Neoplasms; Male; Mice; Mice, Nude; Paclitaxel; Phosphorylation; Pyrimidines; Pyrroles; Recombinant Proteins; Signal Transduction; Transforming Growth Factor alpha; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Thrombospondin-1 (TSP-1), a multifunctional matrix protein, affects tumor growth through modulation of angiogenesis and other stromal biological functions. In several of nine human cancer cell lines derived from liver, brain, pancreas, and bone, expression of TSP-1 was up-regulated in response to the two most representative growth factors, epidermal growth factor (EGF) and transforming growth factor beta1 (TGFbeta1). Expression of TSP-1 was markedly enhanced in hepatic HuH-7 cells by EGF but not by TGFbeta1. In contrast, expression of TSP-1 was markedly enhanced by TGFbeta1, but not by EGF, in osteosarcoma MG63 cells. EGF induced activation of TSP-1 promoter-driven luciferase activity in HuH-7 cells, and the elements between -267 and -71 on the 5' region of TSP-1 gene containing two GC boxes to which Sp1 bound, were found to be responsible for the promoter activation by EGF. However, EGF did not alter TSP-1 mRNA stability in hepatic cells. On the other hand, no such enhancement of the TSP-1 promoter activity by TGFbeta1 appeared in MG63 cells. Enhanced expression of TSP-1 by TGFbeta1 in MG63 cells was partially blocked by exogenous addition of SB203580, an inhibitor of p38 mitogen-activated protein kinase. TGFbeta was found to induce marked elongation of TSP-1 mRNA longevity in osteosarcoma cells when mRNA degradation was assayed in the presence of alpha-amanitin. The up-regulation of TSP-1 by EGF and TGFbeta might play a critical role in modulation of angiogenesis and formation of matrices in tumor stroma.

Topics: Bone Neoplasms; Carcinoma, Hepatocellular; Epidermal Growth Factor; Gene Expression Regulation; Humans; Liver Neoplasms; Osteosarcoma; RNA Stability; Thrombospondin 1; Transcriptional Activation; Transforming Growth Factor beta; Tumor Cells, Cultured; Up-Regulation

Recent data demonstrate that endothelin-1 (ET-1) concentration increases in plasma of men with advanced, hormone-refractory prostate adenocarcinoma. In addition, ET-1 is involved in osteblastic remodelling and new bone formation, suggesting a role for this vasoactive peptide in the metastatic progression of prostate cancer to the bone.. We investigated the regulation of ET-1 expression in androgen-sensitive and insensitive prostate cancer cell lines by androgens and several factors involved in progression of prostate cancer (EGF) and bone remodelling (TGFbeta-1, IL1-alpha and IGF-1).. Northern analysis and radio immunoassay demonstrated that all the ET-1 pathways are tuned off in the androgen-sensitive LNCaP cell line when compared to the androgen-insensitive PC-3 and DU145. In PC-3 cells transfected with a full-length androgen receptor expression vector (PC-3-AR), treatment with androgens reduced gene expression and secretion of ET-1 without affecting the gene expression of ET-3. Collectively, these data support a role for androgens in the regulation of ET-1 production by prostate adenocarcinoma cells. In PC-3 and DU145 cells, ET-1 gene expression and secretion were up-regulated by TGFbeta-1, EGF and IL1-alpha, whereas IGF-1 was ineffective. Conversely, none of the treatments affected ECE-1 or ET-3 gene expression.. In conclusion, ET-1 production by prostate adenocarcinoma cells is down-regulated by androgens and up-regulated by factors involved in tumour progression indicating a role for this peptide in the biology of prostate cancer. In view of the role exerted by ET-1 in the process of bone metastasis, our data suggest the use of ET-1 receptor antagonists in the treatment of advanced prostate cancer.

Topics: Adenocarcinoma; Androgens; Blotting, Northern; Bone Neoplasms; Cytokines; Endothelin-1; Endothelin-3; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Humans; Male; Metalloendopeptidases; Neoplasms, Hormone-Dependent; Prostatic Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Tumor Cells, Cultured

The EWS/FLI-1 fusion gene is characteristic of most cases of Ewing's sarcoma and has been shown to be crucial for tumor transformation and cell growth. In this study we demonstrate a drastic down-regulation of the EWS/FLI-1 protein, and a growth arrest, following serum depletion of Ewing's sarcoma cells. This indicates that growth factor circuits may be involved in regulation of the fusion gene product. Of four different growth factors tested, basic fibroblast growth factor (bFGF) was found to be of particular significance. In fact, upon treatment of serum-depleted cells with bFGF, expression of the EWS/FLI-1 protein and growth of the Ewing's sarcoma cells were restored. In addition, a bFGF-neutralizing antibody, which was confirmed to inhibit FGF receptor (FGFR) phosphorylation, caused down-regulation of EWS/FLI-1. Experiments using specific cell cycle blockers (thymidine and colcemide) suggest that EWS/FLI-1 is directly linked to bFGF stimulation, and not indirectly to cell proliferation. We also demonstrated expression of FGFRs in several tumor samples of Ewing's sarcoma. Taken together, our data suggest that expression of FGFR is a common feature of Ewing's sarcoma and, in particular, that the bFGF pathway may be important for the maintenance of a malignant phenotype of Ewing's sarcoma cells through up-regulating the EWS/FLI-1 protein. Oncogene (2000) 19, 4298 - 4301

Topics: Adenocarcinoma; Antibodies, Monoclonal; Bone Neoplasms; Cell Cycle; Cell Division; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 22; Culture Media, Serum-Free; Demecolcine; Drug Synergism; Epidermal Growth Factor; Fibroblast Growth Factor 2; Fibroblasts; Gene Expression Regulation, Neoplastic; Humans; Insulin-Like Growth Factor I; Male; Neoplasm Proteins; Oncogene Proteins, Fusion; Platelet-Derived Growth Factor; Prostatic Neoplasms; Proto-Oncogene Protein c-fli-1; Receptors, Fibroblast Growth Factor; RNA-Binding Protein EWS; Sarcoma, Ewing; Thymidine; Transcription Factors; Translocation, Genetic; Tumor Cells, Cultured

With the aim of identifying innovative therapeutic strategies for osteosarcoma patients who are refractory to conventional chemotherapy, we analyzed the in vitro effects of the blockage of autocrine circuits. Since the insulin-like growth factor-I receptor (IGF-IR)-mediated loop is relevant to the growth of osteosarcoma, we analyzed the activity of the IGF-IR-blocking antibody alphaIR3 in both sensitive and multidrug-resistant osteosarcoma cell lines. Only limited effects, however, were observed, suggesting the simultaneous existence of other autocrine circuits. Indeed, in a representative panel of 12 human osteosarcoma cell lines, in addition to the IGF-IR-mediated circuit, we demonstrated also a loop mediated by epidermal growth factor receptor as well as the presence of nerve growth factor, low-affinity nerve growth factor receptor as well as tyrosine receptor kinase A in the great majority of osteosarcomas. Therapies based on the inhibition of single circuits may have only limited effects in osteosarcoma, whereas the use of suramin, a drug which, besides other activities, non-selectively interferes with the binding of growth factors to their receptors, appears as a promising alternative, in both sensitive and drug-resistant osteosarcoma cells.

Topics: Antineoplastic Agents; Autocrine Communication; Becaplermin; Bone Neoplasms; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Epidermal Growth Factor; Humans; Insulin-Like Growth Factor I; Insulin-Like Growth Factor II; Neoplasm Proteins; Nerve Growth Factors; Osteosarcoma; Platelet-Derived Growth Factor; Proto-Oncogene Proteins c-sis; Receptor, IGF Type 1; Receptors, Growth Factor; Suramin; Transforming Growth Factor alpha; Tumor Cells, Cultured

Adamantinoma of long bones is a rare bone tumour with (immuno-) histological features of epithelial cells, surrounded by various amounts of osteofibrous tissue. Recent studies have indicated that cells with an epithelial phenotype are most probably the malignant element. There is still debate as to whether the fibrous part should be designed as a benign neoplastic element of a biphasic tumour or as a reactive non-neoplastic tissue next to an epithelioid bone tumour. The expression of fibroblast growth factor type 2 (FGF-2), epidermal growth factor (EGF), and their respective receptors FGFR-1 and EGFR, as well as the proliferation marker Ki-67, was studied in both constituents of adamantinoma in serial sections of 25 cases by immunohistochemistry. Expression of FGF-2 and its receptor was present in both constituents of adamantinoma, but predominated in the epithelial component. Expression of EGF and its receptor was restricted to the epithelial component of adamantinoma. Comparing osteofibrous dysplasia (OFD)-like adamantinoma with classic epithelial cell-rich adamantinoma, the expression of FGF-2, EGF, and EGFR was more intense and in a higher percentage of cells in classic adamantinoma. Proliferative activity was found nearly exclusively in the epithelial component. These data further substantiate the hypothesis that epithelial cells constitute the proliferating tumour cell population responsible for the malignant behaviour of adamantinoma. The data indicate that during progression, the epithelial cells acquire expression of FGF-2, EGF, and EGFR, accompanied by a higher proliferative activity. Within the epithelial cell population, there exists an autocrine pathway of growth stimulation. Furthermore, these data point to an interaction between the epithelial and fibrous components, in which the epithelial cells additionally stimulate fibrous cell growth via a paracrine pathway involving FGF-2.

Topics: Ameloblastoma; Bone Neoplasms; Cell Division; Epidermal Growth Factor; ErbB Receptors; Fibroblast Growth Factor 2; Growth Substances; Humans; Immunoenzyme Techniques; Ki-67 Antigen; Receptors, Growth Factor

Human osteoclasts are well characterized multinucleated cells whose function is the directed resorption of normal bone (NB). Osteoclastic bone destruction accompanies lytic solid tumors and myeloma as well as Paget's disease (PD) of bone and giant cell tumors of bone (GCTB). The mechanism of this stimulation of osteoclastic bone resorption is unknown. This study was designed to detect cytokines present in the multinucleated cells of PD and GCTB in order to determine whether cytokine abnormalities exist to account for bone lysis. Nine cytokines, representing the functions of bone resorption, angiogenesis, tumor necrosis, bone cell proliferation, and osteoblast-osteoclast coupling, were examined by immunohistochemistry using tissue samples from 15 NB, 17 PD, and 19 GCTB patients. Standard nonparametric statistical analysis showed a significant increase (P < 0.01 to 0.05) in immunostaining between osteoclasts of PD and NB for interleukin-6 (Il-6), tumor necrosis factor beta (TNFbeta), epidermal growth factor (EGF), platelet derived growth factor (PDGF), and basic fibroblast growth factor (bFGF). There was a statistically significant decrease in immunostaining of giant cells of GCTB as compared with NB for transforming growth factor beta (TGFbeta), but no other differences from normal osteoclasts. The increase in staining of PD osteoclasts over the giant cells of GCTB was significant (P < 0.01) for Il-6, TNFbeta, PDGF, bFGF and insulin growth factor-1 (IGF-1), and (P < 0. 05) for Il-1 and EGF. It was concluded that marked cytokine differences exist in vivo between osteoclasts of NB and PD lesions consistent with stimulated resorption. Alternatively, "osteoclastoma" cells in the center of the tumor did not overexpress the cytokines associated with bone lysis, suggesting some other mechanism for stimulated resorption.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Bone Neoplasms; Bone Resorption; Child; Child, Preschool; Cytokines; Epidermal Growth Factor; Female; Fibroblast Growth Factor 1; Fibroblast Growth Factor 2; Giant Cell Tumor of Bone; Giant Cells; Humans; Insulin-Like Growth Factor I; Interleukin-1; Interleukin-6; Lymphotoxin-alpha; Male; Middle Aged; Osteitis Deformans; Osteoclasts; Platelet-Derived Growth Factor; Transforming Growth Factor beta

We investigated the characteristics of nucleolar organizer regions (NORs) and epidermal growth factor receptor (EGFR) on differentiated Dunn osteosarcoma in response to dibutyryl cyclic adenosine 3',5'-monophosphate (Bt2cAMP). In the presence of 3 mM Bt2cAMP, the mean number of NORs was significantly decreased in 24 hrs and 48 hrs compared with the untreated group. Also, EGFR affinity was decreased and the EGFR number was increased in response to the cellular differentiation by Bt2cAMP. The decrease in EGFR affinity might be considered as an indicator of differentiation or the mature state of the cells. Thus, the present study provides a new clue to support differentiation of osteosarcoma cells from the viewpoint of NORs findings and EGFR analysis as a differentiation marker.

Topics: Animals; Biomarkers, Tumor; Bone Neoplasms; Bucladesine; Cell Differentiation; DNA, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Female; Iodine Radioisotopes; Mice; Mice, Inbred C3H; Nucleolus Organizer Region; Osteosarcoma; Silver Staining; Tumor Cells, Cultured

Prostate tumor cells preferentially metastasize to bony sites and lymph nodes at a frequency in excess of that which would be predicted by random tumor cell dissemination. In order to determine whether chemoattractants in these organs promote organ-specific metastasis, we utilized human cell lines derived from and/or related to these organs as sources of potential chemoattractants. Secretory proteins derived from the cell lines MG-63 (osteosarcoma), SK-ES-1 (Ewing's sarcoma), and KG-1 (leukemia) stimulated chemomigration of the TSU-pr1 prostate tumor cells in a dose-dependent manner in Boyden chambers. In addition, secretory proteins from a human prostatic stromal cell line (hPS) and from the TSU-Pr1 prostate tumor cell line were also able to stimulate chemomigration of the TSU-pr1 cells through Boyden chambers. Since lymph nodes and bony sites represent organs of hematopoietic/lymphoid proliferation and activation, we undertook identification of specific cytokines present at these sites which may promote the chemomigration of prostate tumor cells. In this context, the cytokines interleukin-1 alpha, interleukin-2, interleukin-6, tumor necrosis factor-beta, transforming growth factor-beta, interferon alpha 2-a, and granulocyte-macrophage colony-stimulating factor did not stimulate chemomigration of the TSU-pr1 prostate tumor cell line. In contrast, the cytokine epidermal growth factor (EGF) stimulated chemomigration of the TSU-pr1 prostate tumor cells through the Boyden chambers in a dose-dependent manner. Western blot analysis of secretory proteins from the cell lines KG-1, SK-ES-1, MG-63, hPS, and TSU-pr1 identified EGF-immunoreactive proteins in all cases. In addition, EGF immunoreactivity was localized to the stroma of the human prostate, the osteogenic stroma of pelvic medullary bone, and the stroma within the capsule and trabeculae of pelvic lymph nodes. Hence, these results demonstrate that the cytokine EGF promotes the chemomigration of the TSU-pr1 prostate tumor cell line, and that EGF within the stroma of pelvic lymph nodes and medullary bone may act as a chemoattractant for prostate tumor cells, thereby facilitating the preferential formation of metastatic foci within these organs.

Topics: Bone and Bones; Bone Neoplasms; Chemotactic Factors; Chemotaxis; Cytokines; Dose-Response Relationship, Drug; Epidermal Growth Factor; Fluorescent Antibody Technique; Humans; Immunoblotting; Lymph Nodes; Lymphatic Metastasis; Male; Pelvis; Prostate; Prostatic Neoplasms; Tumor Cells, Cultured

Androgens are required for the optimal growth and development of both the normal prostate and steroid-sensitive prostate cancer. PC3 prostate cancer cell lines stably expressing the human androgen receptor (AR) and possessing an androgen-sensitive phenotype (PC3-hAR) were used to examine the role of the epidermal growth factor receptor (EGFR) in androgen-stimulated prostate cancer cell growth. Epidermal growth factor (EGF) and dihydrotestosterone (DHT) independently induced the growth of PC3-hAR cells. Moreover, EGF and DHT in combination exerted a synergistic effect on PC3-hAR cell growth. DHT-exposed PC3-hAR cells expressed a greater than 2-fold increase in EGFR mRNA and 50% more EGFR protein than controls. Time course radioligand-binding assays confirmed these findings by showing an elevation in EGF binding in the DHT-exposed PC3-hAR cells. In addition, radioligand competition-binding studies revealed a 2-fold increase in EGFR-EGF binding affinity in the PC3-hAR cells after DHT treatment. However, no enhancement of transforming growth factor alpha or EGF expression was detected because DHT did not affect the levels of these cytokines in the PC3-hAR cell lysate or conditioned media. Our observations suggest that DHT increases both EGFR number and receptor-ligand affinity in androgen-sensitive prostate cancer cells and that these effects correlate with increased EGF binding and an enhanced mitogenic response to EGF.

Topics: Adenocarcinoma; Androgens; Animals; Bone Neoplasms; Cell Division; Dihydrotestosterone; Drug Synergism; Epidermal Growth Factor; ErbB Receptors; Gene Expression; Gene Expression Regulation, Neoplastic; Humans; Lymphatic Metastasis; Male; Mice; Neoplasms, Hormone-Dependent; Prostatic Neoplasms; Receptors, Androgen; Stimulation, Chemical; Transforming Growth Factor alpha; Tumor Cells, Cultured; Up-Regulation

Interleukin-1 (IL-1) is a potent bone resorbing cytokine with diverse biological effects. We previously reported that IL-1 inhibits PDGF-AA-induced biological activities including PDGF-AA-induced tyrosyl phosphorylation. In the present studies, we first investigated and compared the tyrosyl phosphorylation pattern induced by EGF, IGF-1, PDGF-AA, and bFGF in human osteoblastic cells. We then examined the effect of IL-1 on the tyrosyl phosphoproteins induced by each ligand. Immunoblot analyses show that EGF, IGF-1, and PDGF-AA each elicit a different pattern of tyrosyl phosphorylated proteins in normal human osteoblastic cells. IL-1 beta inhibits PDGF-AA induced autophosphorylation by down-regulation of the PDGF-alpha receptor, as demonstrated by immunoprecipitation experiments. For other ligand-induced tyrosyl phosphoproteins, IL-1 beta reduced the intensity of EGF-induced pp55,000, and IGF-1 induced pp185,000 and pp175,000. These experiments indicate that IL-1 inhibits phosphorylation of specific proteins induced by growth factors. By using inhibitors of secondary message pathways, we determined that the inhibitory effect of IL-1 beta on PDGF-AA receptor binding and receptor tyrosyl autophosphorylation was not dependent on protein kinase A, protein kinase C, or the formation of prostaglandins. These data suggest the existence of an alternative pathway that may participate in IL-1 beta signaling.

Topics: Bone Neoplasms; Bone Resorption; Cell Division; Electrophoresis, Polyacrylamide Gel; Epidermal Growth Factor; Fibroblast Growth Factor 2; Fibroblasts; Humans; Interleukin-1; Osteoblasts; Osteosarcoma; Phosphorylation; Platelet-Derived Growth Factor; Proteins; Receptors, Growth Factor; Receptors, Platelet-Derived Growth Factor; Signal Transduction; Tumor Cells, Cultured; Tyrosine

The expression of both epidermal growth factor (EGF) and platelet-derived growth factor (PDGF), and of their receptors (EGFR and PDGFR) was immunohistochemically examined in 37 cases of osteosarcoma. Furthermore, immunostaining for p53 protein and Ki-67 antigen by MIB-1 was carried out and compared with the above results. EGFR (81%) expressed more often than EGF (51%) and the expression of EGF and EGFR, and PDGF and PDGFR were recognized in 49% and 38%, respectively. In eleven cases (30%), the expression of both growth factors and their receptors was combined. Anaplastic osteosarcoma showed higher MIB-1 index than osteoblastic and fibroblastic subtypes (P < 0.05). High grade osteosarcomas (G3 and G4) revealed higher MIB-1 index compared with low grade tumors (G1 and G2). PDGF positive tumors (MIB-1 index: 20.0) showed significantly higher proliferation compared with PDGF negative tumors (MIB-1 index: 6.5) (P < 0.01). Five out of 37 cases (13.5%) showed positive immunoreaction for p53. There was no correlation of p53 status with MIB-1 index and the expression of growth factors or their receptors. Our results suggest that PDGF expression may be an important mediator of cell proliferation control, via an autocrine mechanism, in human osteosarcoma.

Topics: Antibodies, Monoclonal; Bone Neoplasms; Cell Division; Epidermal Growth Factor; ErbB Receptors; Growth Substances; Humans; Immunohistochemistry; Osteosarcoma; Platelet-Derived Growth Factor; Receptors, Platelet-Derived Growth Factor; Tumor Suppressor Protein p53

Sequence information for aspartyl beta-hydroxylase (AspH), which specifically hydroxylates one Asp or Asn residue in certain epidermal growth factor (EGF)-like domains of a number of proteins, is so far only described for bovine species. We have isolated a 4.3-kb cDNA encoding the human AspH (hAspH) by immunoscreening of a human osteosarcoma (MG63) cDNA library in lambda ZAP with an antiserum raised against membrane fractions of these cells. Northern blot analyses revealed two transcripts with lengths of 2.6 and 4.3 kb. The deduced amino acid (aa) sequence of this cDNA encodes a protein of 757 aa (85 kDa). Comparison with the deduced bovine AspH (bAspH) aa sequence showed striking differences in the N-terminal portion of this protein. In vitro transcription and translation in the presence of canine pancreas microsomes yielded a 56-kDa protein. Western blot analyses of membrane fractions from MG63 cells with AspH-specific antibodies revealed a protein of the same M(r). These results suggest a posttranslational cleavage of the catalytic C terminus in the lumen of the endoplasmic reticulum.

Topics: Amino Acid Sequence; Animals; Base Sequence; Blotting, Northern; Bone Neoplasms; Cattle; Cell Membrane; Cloning, Molecular; Epidermal Growth Factor; Hominidae; Humans; Immune Sera; Mixed Function Oxygenases; Molecular Sequence Data; Osteosarcoma; Protein Biosynthesis; Sequence Homology, Amino Acid; Transcription, Genetic; Tumor Cells, Cultured

Prostaglandin E2 (PGE2), PTH, and epidermal growth factor (EGF) are potent regulators of osteoblast proliferation. In UMR 106-01 rat osteosarcoma cells with osteoblast-like features, PGE2 and PTH inhibit, while EGF stimulates, mitogenesis. Both PGE2 and PTH increase intracellular cAMP levels, cytosolic calcium, and inositol phosphate turnover. In a variety of cell types, EGF mediates its effects in part via activation of receptor protein-tyrosine kinase and other protein kinases, such as protein kinase-C. The nuclear mechanisms of PGE2, PTH, and EGF regulation of osteoblast proliferation are unknown. Accordingly, we have examined the effects of these agents on mitogenesis, second messenger generation, and primary response genes, which may link second messenger activation to subsequent alterations in gene expression. Northern blot analysis of mRNA from UMR 106-01 cells treated for 3 h with 2 microM PGE2, 10 nM PTH, or 10 ng/ml EGF in the presence of cycloheximide demonstrated that all three agents induced the expression of c-fos and c-jun mRNA. In contrast, only EGF stimulated cellular proliferation and induced Egr-1 mRNA. Also, unlike PGE2 and PTH, EGF did not increase intracellular cAMP levels. c-fos mRNA was induced by treatment with 50 ng/ml tetradecanoyl phorbol acetate or by 40 ng/ml forskolin, while induction of Egr-1 mRNA was stimulated by treatment with tetradecanoyl phorbol acetate, but not forskolin. Thus, EGF signal transduction differs from that of PGE2 and PTH in UMR 106-01 osteoblast-like cells, in that EGF does not stimulate the protein kinase-A second messenger system, but causes activation of Egr-1, a primary response gene that may play a role in the mitogenic effect of EGF.

Topics: Animals; Bone Neoplasms; Calcium; Colforsin; Cyclic AMP; Cycloheximide; Dinoprostone; DNA-Binding Proteins; Early Growth Response Protein 1; Enzyme Activation; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Genes, fos; Genes, jun; Immediate-Early Proteins; Inositol Phosphates; Mitosis; Osteoblasts; Osteosarcoma; Parathyroid Hormone; Protein Kinases; Radioimmunoassay; Rats; RNA, Messenger; Signal Transduction; Tetradecanoylphorbol Acetate; Thymidine; Transcription Factors; Tritium; Tumor Cells, Cultured; Zinc Fingers

Malignant prostatic carcinoma, a major cause of cancer mortality in males, most often metastasizes to secondary sites in bone. Frequently, the growth rate of the secondary tumor in bone marrow is considerably greater than that of the slowly growing primary prostatic tumor. We now report that two lines of human prostatic carcinoma cells proliferate in response to conditioned media from unstimulated human, rat, or bovine bone marrow. Nonprostatic tumor cell lines showed little or no growth response to the same medium. The proliferative activity found in bone marrow was not duplicated by any of a variety of purified growth factors including epidermal growth factor (EGF), acidic or basic fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), transforming growth factor (TGF) alpha or beta, interleukins 1, 2, 3, 4 or 6, granulocyte (G), macrophage (M) or granulocyte-macrophage (GM) colony stimulating factor (CSF). Whereas a mixture of G-CSF, M-CSF, and IL 3 produced a mitogenic response in the prostatic carcinoma cells, these three factors were not present in our bone marrow samples in sufficient quantities to promote the observed proliferative response. To further identify the cellular source of the proliferative activity present in bone marrow-conditioned medium, we tested conditioned media made from human bone marrow stromal cells. The stromal cell conditioned medium stimulated increased growth of the prostatic carcinoma cells to levels equivalent to those observed with the bone marrow conditioned medium. These results suggest that novel mitogenic factors that are produced by bone marrow stromal cells and remain in the bone marrow cavity may account, in part, for the preferential growth of prostatic metastases in bone.

Topics: Adenocarcinoma; Animals; Bone Marrow; Bone Marrow Cells; Bone Neoplasms; Cattle; Cell Line; Colony-Stimulating Factors; Culture Media; Epidermal Growth Factor; Fibroblast Growth Factors; Humans; Male; Platelet-Derived Growth Factor; Prostatic Neoplasms; Rats; Transforming Growth Factors