Differential effects of cetuximab and AEE 788 on epidermal growth factor receptor (EGF-R) and vascular endothelial growth factor receptor (VEGF-R) in thyroid cancer cell lines.

This study evaluated the role of EGF and the effects of EGF-targeting drugs (Cetuximab, AEE 788) on growth, apoptosis, and autocrine VEGF-secretion of thyroid cancer (TC) cells. Autocrine activation of the epidermal growth factor receptor (EGF-R) is commonly regarded to contribute to the malignant phenotype of TC cells and may therefore represent a rational therapeutic target. Out of a number of TC cell lines two anaplastic (Hth74, C643), one follicular (FTC133), and one papillary thyroid cancer cell line (TPC1) were analyzed in depth for VEGF-R-and EGF-R-expression, basal and EGF-stimulated (1-100 ng/ml) VEGF protein secretion and proliferation. Subsequently the antiprolifereative and antiangiogenic effect of cetuximab (Erbitux), a monoclonal antibody that blocks the EGF-R and AEE 788, a novel dual-kinase inhibitor of EGF-R and VEGF-R were assessed, and the downstream EGF-R signal transduction was analyzed by means of detecting phosphorylated pEGF-R, pVEGF-R, pAkt, and p-MAPK. EGF stimulated VEGF-mRNA expression and protein secretion in all TC cell lines. The EGF-R antagonist Cetuximab consistently decreased VEGF secretion in all TC cell lines (min. 15%, n.s. in C643 cells and max. 90% in Hth74 cells, P < 0.05), but did not affect tumor cell proliferation in vitro. In contrast, the EGF-R- and VEGF-R-kinase inhibitor AEE 788 not only reduced VEGF secretion (min. 55%, P < 0.05 in C643 and max. 75%, P < 0.05, in FTC133), but also exhibited a dose-dependent inhibition of tumor cell proliferation (min. 75%, P < 0.05 in C643 and max. 95%, P < 0.05 in Hth74) and was a potent inductor of apoptosis in two of four TC cell lines. These effects were always accompanied by reduced levels of pEGF-R, pVEGF-R, pAkt, and pMAPK. Although inhibition of the EGF-receptor by Cetuximab potently disrupts autocrine secretion of VEGF, only the concurrent inhibition of the VEGF- and EGF receptor, e.g., by AEE 788 induces reduced proliferation and apoptosis in vitro. This suggests a particular rationale for the use of tyrosine kinase inhibitors with dual modes of action such as AEE 788 in thyroid cancer.

Functional thyrotropin receptor attenuates malignant phenotype of follicular thyroid cancer cells.

Thyrotropin (TSH) is a thyroid-specific growth factor inducing differentiated function and growth of thyrocytes in vitro. In thyroid cancer, loss of TSH-receptor (TSHR) expression is a sign of de-differentiation and is believed to contribute to the malignant phenotype. The present studies aimed to determine the in vitro and in vivo effects of functioning TSHR in the follicular thyroid cancer cell line HTC, a subclone of FTC133 cells, lacking endogenous expression of TSHR, and HTCtshr+ cells transfected with human TSHR-cDNA. HTCtshr+ cells grew faster in vitro (doubling time 1.15 vs 1.56 d, p < 0.05) and TSH caused a dose-dependent growth response. Adhesion to and invasion through reconstituted basement membrane were reduced in HTCtshr+ cells, but when stimulated with TSH increased to levels comparable to naïve HTC cells. In vivo, tumor latency was 11 d for naïve HTC as compared to 21 d for HTCtshr+ xenografts. Smaller tumor volumes were registered for HTCtshr+ cells (250 +/- 217 vs 869 +/- 427 mm3, p < 0.05). Angiogenesis, as determined by vascular surface density (VSD) of experimental tumors, was enhanced in naïve HTC tumors (VSD 0.87 +/- 0.1 microm-1 vs 0.55 +/- 0.2 microm-1 in HTCtshr+, p < 0.05). VEGF secretion was more pronounced in naïve HTC cells stimulated with EGF, than in HTCtshr+ cells stimulated with either TSH or EGF. In conclusion, regained expression of functional TSHR in the follicular thyroid cancer cell line HTC alters in vitro features commonly associated with the malignant phenotype. Smaller tumors and reduced angiogenesis of xenotransplanted HTC cells with functioning TSHR suggest a less aggressive in vivo phenotype. The present data highlight the pivotal role of TSHR to affect transformed thyrocytes in vitro and in vivo. They also suggest a role for EGF as a modulator of angiogenesis in thyrocytes devoid of TSHR.

Targeting the EGF/VEGF-R system by tyrosine-kinase inhibitors--a novel antiproliferative/antiangiogenic strategy in thyroid cancer.

AIM: In thyroid cancer (TC), endothelial growth factor (EGF) has been associated with dedifferentiation, tumor cell proliferation, and angiogenesis. Vascular endothelial growth factor (VEGF) has been documented to be the main stimulator of angiogenesis in the thyroid gland. Patients with undifferentiated thyroid cancer are in desperate need of new therapeutic strategies because common protocols of therapy usually fail. The aim of this study, therefore, was to evaluate two tyrosine-kinase inhibitors (TKI, ZD 1839 gefitinib and ZD 6474 vandetanib), directed against the EGF/VEGF receptor for possible antitumor therapy in thyroid cancer. METHODS: EGF/VEGF-R was documented in anaplastic (Hth74, C643), follicular (FTC133), and papillary (TPC1) thyroid cancer cell lines by Western blot analysis. The antiproliferative effect of two TKI (0.1-10 microM) on thyroid cancer cell lines in vitro was quantified by MTT assay, the antiangiogenic effect by assessing secretion of VEGF by enzyme-linked immunosorbent assay (R&D Systems). ZD 1839 is mainly directed against EGF-R and ZD 6474 against VEGF-R (AstraZeneca, UK), single applications and combinations of compounds were evaluated. RESULTS: EGF-R and VEGF-R as well as the phosphorylated receptor were documented in all of the cell lines. Administration of ZD1839 led to an up to 90% reduction of cell number in Hth74, 80% in C643, 50% in FTC133, and 90% in TPC1 (p < 0.05). ZD1839 induced a decrease of VEGF secretion between 30% in C643 and 90% in Hth74. Administration of ZD6474 led to an up to 95% reduction of cell number in Hth74, 85% in C643, 90% in FTC133, and 90% in TPC1 (p < 0.05). The ZD6474 induced decrease of VEGF secretion ranged between 20% (FTC133) and 60% (TPC1). Combinations of IC50 concentrations of TKI showed synergistic effects, resulting in additional inhibition of proliferation between 50 and 90% compared to single drug administration. CONCLUSION: The EGF/EGF-R system resembles a powerful VEGF-stimulating pathway in all histiotypes of TC and can be inhibited by TKI. TKI directed against EGF-R as well as VEGF-R inhibit tumor cell proliferation and VEGF secretion in vitro. Combinations of TKI are more effective than strategies using single agents. It is suggested that targeting EGF-R/VEGF-R-mediated pathways may have therapeutic potential in some undifferentiated thyroid cancers.

Differential pattern of integrin receptor expression in differentiated and anaplastic thyroid cancer cell lines.

Adhesion of tumor cells to the extracellular matrix (ECM) is a crucial step for the development of metastatic disease and is mediated by specific integrin receptor molecules (IRM). The pattern of metastatic spread differs substantially among the various histotypes of thyroid cancer (TC). However, IRM have only occasionally been characterized in TC until now. IRM expression was investigated in 10 differentiated (FTC133, 236, 238, HTC, HTC TSHr, XTC, PTC4.0/4.2, TPC1, Kat5) and two anaplastic TC cell lines (ATC, C643, Hth74), primary cultures of normal thyroid tissue (Thy1,3), and thyroid cancer specimens (TCS). Expression of 16 IRM (beta1-4, beta7, alpha1-6, alphaV, alphaIIb, alphaL, alphaM, alphaX) and of four IRM heterodimers (alpha2beta1, alpha5beta1, alphaVbeta3, alphaVbeta5), was analyzed by fluorescent-activated cell sorter (FACS) and immunohistochemical staining. Thyroid tumor cell adhesion to ECM proteins and their IRM expression in response to thyrotropin (TSH) was assessed. Follicular TC cell lines presented high levels of integrins alpha2, alpha3, alpha5, beta1, beta3 and low levels of alpha1, whereas papillary lines expressed a heterogenous pattern of IRM, dominated by alpha5 and beta1. ATC mainly displayed integrins alpha2, alpha3, alpha5, alpha6, beta1 and low levels of alpha1, alpha4 and alphaV. Integrin heterodimers correlated with monomer expression. Evaluation of TCS largely confirmed these results with few exceptions, namely alpha4, alpha6, and beta3. The ability of TC cell lines to adhere to purified ECM proteins correlated with IRM expression. TSH induced TC cell adhesion in a dose-dependent fashion, despite an unchanged array of IRM expression or level of a particular IRM. Thyroid carcinoma cell lines of different histogenetic background display profoundly different patterns of IRM expression that appear to correlate with tumor aggressiveness. In vitro adhesion to ECM proteins and IRM expression concur. Finally, TSH-stimulated adhesion of thyroid tumor cell lines to ECM may not be associated with altered IRM expression.

Activation and proliferation signals in murine macrophages: synergistic interactions between the hematopoietic growth factors and with phorbol ester for DNA synthesis.

There has been recent interest in the synergistic interactions between the growth factors involved in the in vitro control of hematopoiesis and other cell lineages. As a convenient model system, such interactions governing the DNA synthesis in murine bone marrow-derived macrophages (BMMs) were studied. By themselves, murine colony-stimulating factor-1 (CSF-1) and recombinant murine granulocyte-macrophage CSF (GM-CSF) were stimulators of DNA synthesis in quiescent or noncycling BMMs, whereas recombinant murine interleukin-3 (IL-3) and the phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), were weak mitogens. On the other hand, murine granulocyte CSF (G-CSF), concanavalin A (Con A), and lipopolysaccharide (LPS) were inactive on their own. When the quiescent BMMs were exposed to combinations of the CSFs, there were striking synergistic effects for both GM-CSF and IL-3 with suboptimal doses of CSF-1, with a smaller effect for GM-CSF with IL-3 and little or no effect for CSF-1 with G-CSF. CSF-1, GM-CSF, and IL-3 could also synergize with TPA; CSF-1 cooperated with 1-oleoyl-2-acetylglycerol (OAG), both sets of results pointing to an interaction with protein kinase C. LPS completely abolished the CSF-1-mediated stimulation of DNA synthesis. We propose that BMMs are suitable normal cells in which to examine in depth the various mechanistic possibilities for these interactions.

Activation and proliferation signals in murine macrophages: stimulation of glucose uptake by hemopoietic growth factors and other agents.

Purified colony-stimulating factor (CSF-1) (or macrophage colony stimulating factor [M-CSF]) stimulated the glucose uptake of murine bone marrow-derived macrophages (BMM) and resident peritoneal macrophages (RPM) as measured by 3H-2-deoxyglucose (2-DOG) uptake. Similar concentrations of CSF-1 stimulated the 2-DOG uptake and DNA synthesis in BMM. Other purified hemopoietic growth factors, granulocyte-macrophage CSF (GM-CSF) and interleukin-3 (IL-3) (or multi-CSF), and the tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), even though differing in their mitogenic capabilities on BMM, were also stimulators of 2-DOG uptake in BMM and RPM. The nonmitogenic agents, lipopolysaccharide (LPS) and concanavalin A (Con A), were also active. The inhibition by cytochalasin B and by high concentrations of D-glucose suggest that the basal and stimulated 2-DOG uptake occurred via a carrier-facilitated D-glucose transport system. The responses of the two macrophage populations to the hemopoietic growth factors and to the other agents were quite similar, suggesting that events that are important for the induction of DNA synthesis are not tightly coupled to the earlier rise in glucose uptake. For the BMM, the ability of a particular agent to stimulate glucose uptake did not parallel its ability to promote cell survival. However, stimulation of glucose uptake could still be a necessary but insufficient early macrophage response for cell survival and subsequent DNA synthesis.

CSF-1 stimulates glucose uptake in murine bone marrow-derived macrophages.

3H-2-deoxyglucose was used as an isotopic tracer for the measurement of glucose uptake into quiescent murine bone marrow derived macrophages. A purified colony stimulating factor (CSF-1) was shown to stimulate 3H-2-deoxyglucose uptake in a dose-dependent manner. This stimulation was rapid, with a maximal effect seen at 20-30 minutes after growth factor addition. Both the inhibition by cytochalasin B and also the relative degree of competition by high concentrations of a series of glucose analogues suggest that the basal and CSF-1 stimulated 2-deoxyglucose uptake occur via a carrier facilitated D-glucose transport system. The data indicate that a purified growth factor can increase the glucose uptake in macrophages, a finding which could be relevant to the survival and/or the proliferative response of this and other haemopoietic cell types.

The stimulation of human synovial fibroblast plasminogen activator activity. Involvement of cyclic AMP and cyclooxygenase products.

The plasminogen activator activity of human synovial fibroblasts is raised by a monocyte-derived polypeptide, synovial activator and also by all-trans retinoic acid. The elevation of the synovial cell plasminogen activator activity by the two stimuli is potentiated both by agents which can raise cellular cyclic AMP levels, namely prostaglandin E2, cholera toxin and 3-isobutyl-1-methylxanthine, and also by exogenous 8-bromocyclic AMP. These findings suggest that there might be a substrate, which is phosphorylated by a cyclic AMP-dependent protein kinase and which is important in the modulation of the synovial cell plasminogen activator activity by the two stimuli. Prostanoids can be important in the stimulation of the synovial fibroblast plasminogen activator activity by mononuclear cell supernatants, since indomethacin can inhibit the increase in proteinase activity.

Regulation of plasminogen activator production by bone-resorbing hormones in normal and malignant osteoblasts.

The plasminogen activator (PA) activity of clonal rat osteogenic sarcoma cell (phenotypically osteoblast) and of osteoblast-rich rat calvarial cells is shown to be increased by treatment with the bone-resorbing hormones, PTH, 1,25-dihydroxyvitamin D3, prostaglandin E2, and epidermal growth factor. Dose-dependent increases were observed, after a lag period of 4 to 8 h. Stimulated and control PA activities were inhibited by actinomycin D and cycloheximide but not by cytosine arabinoside. Glucocorticoid hormones prevented the hormone stimulation, but other steroids did not. Calcitonin had no effect either on basal or on hormone-treated PA activity. Isobutyl-methylxanthine alone increased PA activity and enhanced responsiveness to PTH and to prostaglandin E2. These data point to a common pathway in the actions upon osteoblasts of several hormones with diverse initial cellular actions and raise the possibility that the PA/plasmin system may contribute to cellular mechanisms of bone turnover.

Stimulation of plasminogen activator in osteoblast-like cells by bone-resorbing hormones.

Hormonal control of plasminogen activator (PA) was studied in clonal rat osteogenic sarcoma cells which are phenotypically osteoblast, and in osteoblast-rich rat bone cell cultures. The bone-resorbing hormones (parathyroid hormone, prostaglandin E2, epidermal growth factor and 1,25-dihydroxyvitamin D3) stimulated PA activity in both cell types. The relative efficacies of vitamin D metabolites and of prostanoids reflect their relative potencies as stimulators of bone resorption.