FTY720, a potent immunomodulator, becomes phosphorylated in vivo (FTY-P) and interacts with sphingosine-1-phosphate (S1P) receptors. Recent studies showed that FTY-P affects vascular endothelial growth factor (VEGF)-induced vascular permeability, an important aspect of angiogenesis. We show here that FTY720 has antiangiogenic activity, potently abrogating VEGF-and S1P-induced angiogenesis in vivo in growth factor implant and corneal models. FTY720 administration tended to inhibit primary and significantly inhibited metastatic tumor growth in a mouse model of melanoma growth. In combination with a VEGFR tyrosine kinase inhibitor PTK787/ ZK222584, FTY720 showed some additional benefit. FTY720 markedly inhibited tumor-associated angiogenesis, and this was accompanied by decreased tumor cell proliferation and increased apoptosis. In transfected HEK293 cells, FTY-P internalized S1P 1 receptors, inhibited their recycling to the cell surface, and desensitized S1P receptor function. Both FTY720 and FTY-P apparently failed to impede VEGF-produced increases in mitogen-activated protein kinase activity in human umbilical vascular endothelial cells (HUVEC), and unlike its activity in causing S1PR internalization, FTY-P did not result in a decrease of surface VEGFR2 levels in HUVEC cells. Pretreatment with FTY720 or FTY-P prevented S1P-induced Ca 2+ mobilization and migration in vascular endothelial cells. These data show that functional antagonism of vascular S1P receptors by FTY720 potently inhibits angiogenesis; therefore, this may provide a novel therapeutic approach for pathologic conditions with dysregulated angiogenesis.
We investigated the significance of erythropoietin receptor (EPOR) expression following treatment with recombinant human erythropoietin (rHuEPO; epoetin A) and the effect of recombinant epoetins (epoetin A, epoetin B, and darbepoetin A) alone or in combination with anticancer therapy on tumor growth in two well-established preclinical models of breast carcinoma (MDA-MB-231 and MCF-7 cell lines). Expression and localization of EPOR under hypoxic and normoxic conditions in MDA-MB-231 and MCF-7 cells were evaluated by immunoblotting, flow cytometry, and immunohistochemistry. EPOR binding was evaluated using [125 I]rHuEPO. Proliferation, migration, and signaling in MDA-MB-231 and MCF-7 cells following treatment with rHuEPO were evaluated. Tumor growth was assessed following administration of recombinant epoetins alone and in combination with paclitaxel (anticancer therapy) in orthotopically implanted MDA-MB-231 and MCF-7 breast carcinoma xenograft models in athymic mice. EPOR expression was detected in both tumor cell lines. EPOR localization was found to be exclusively cytosolic and no specific [ 125 I]rHuEPO binding was observed. There was no stimulated migration, proliferation, or activation of mitogen-activated protein kinase and AKT following rHuEPO treatment. In mice, treatment with recombinant epoetins alone and in combination with paclitaxel resulted in equivalent tumor burdens compared with vehicle-treated controls. Results from our study suggest that although EPOR expression was observed in two well-established breast carcinoma cell lines, it was localized to a cytosolic distribution and did not transduce a signaling cascade in tumors that leads to tumor growth. The addition of recombinant epoetins to paclitaxel did not affect the outcome of paclitaxel therapy in breast carcinoma xenograft models. These results show that recombinant epoetins do not evoke a physiologic response on EPOR-bearing tumor cells as assessed by numerous variables, including growth, migration, and cytotoxic challenge in preclinical in vivo tumor models. [Mol Cancer Ther 2006;5(2):347 -55]
Cell cycle kinase inhibitors have advanced into clinical trials in oncology. One such molecule, JNJ-7706621, is a broad-spectrum inhibitor of the cyclin-dependent kinases and Aurora kinases that mediate G 2 -M arrest and inhibits tumor growth in xenograft models. To determine the putative mechanisms of resistance to JNJ-7706621 that might be encountered in the clinic, the human epithelial cervical carcinoma cell line (HeLa) was exposed to incrementally increasing concentrations of JNJ-7706621. The resulting resistant cell population, designated HeLa-6621, was 16-fold resistant to JNJ-7706621, cross-resistant to mitoxantrone (15-fold) and topotecan (6-fold), and exhibited reduced intracellular drug accumulation of JNJ-7706621. ABCG2 was highly overexpressed at both the mRNA (f163-fold) and protein levels. The functional role of ABCG2 in mediating resistance to JNJ-7706621 was consistent with the following findings: (a) an ABCG2 inhibitor, fumitremorgin C, restored the sensitivity of HeLa-6621 cells to JNJ-7706621 and to mitoxantrone; (b) human embryonic kidney-293 cells transfected with ABCG2 were resistant to both JNJ-7706621 and mitoxantrone; and (c) resistant cells that were removed from the drug for 12 weeks and reverted to susceptibility to JNJ-7706621 showed near-normal ABCG2 RNA levels. ABCG2 is likely to limit the bioavailability of JNJ-7706621 because oral administration of JNJ-7706621 to Bcrp (the murine homologue of ABCG2) knockout mice resulted in an increase in the plasma concentration of JNJ-7706621 compared with wild-type mice. These findings indicate that ABCG2 mediates the resistance to JNJ-7706621 and alters the absorption of the compound following administration.
JNJ-28871063 is a potent and highly selective pan-ErbB kinase inhibitor from a novel aminopyrimidine oxime structural class that blocks the proliferation of epidermal growth factor receptor (EGFR; ErbB1)-and ErbB2-overexpressing cells but does not affect the growth of non-ErbB-overexpressing cells. Treatment of human cancer cells with JNJ-28871063 inhibited phosphorylation of functionally important tyrosine residues in both EGFR and ErbB2 and blocked downstream signal transduction pathways responsible for proliferation and survival. A single dose of compound reduced phosphorylation of ErbB2 receptors in tumor-bearing mice, demonstrating target suppression in vivo. Tissue distribution studies show that JNJ-28871063 crosses the blood-brain barrier and penetrates into tumors, where it is able to accumulate to higher levels than those found in the plasma. JNJ-28871063 showed oral antitumor activity in human tumor xenograft models that overexpress EGFR and ErbB2. In an intracranial ErbB2-overexpressing tumor model, JNJ-28871063 extended survival relative to untreated animals. The brain is a primary site of metastasis for EGFR-overexpressing lung cancers and ErbB2-overexpressing breast cancers. Therefore, the ability to penetrate into the brain could be an advantage over existing therapies such as trastuzumab (Herceptin) and cetuximab (Erbitux), which are antibodies and do not cross the blood-brain barrier. These results show that JNJ-28871063 is orally bioavailable, has activity against EGFR and ErbB2-dependent tumor xenografts, and can penetrate into the brain and inhibit ErbB2-overexpressing tumor growth.The epidermal growth factor receptor (EGFR; HER1; ErbB1) and ErbB2 (HER2; neu) are prototypic members of the ErbB family of tyrosine kinase receptors that also includes ErbB3 (HER3) and ErbB4 (HER4). Multiple ligands can bind to and activate ErbB receptors, inducing autophosphorylation, dimerization, and the phosphorylation of cellular substrates that mediate increased DNA synthesis, cell proliferation, angiogenesis, metastasis, and resistance to apoptosis (reviewed in Hynes and Lane, 2005 ABBREVIATIONS: EGFR, human epidermal growth factor receptor; EGF, epidermal growth factor; ErbB2, human epidermal growth factor receptor-2; HER, human epidermal growth factor receptor; JNJ-28871063, 4-amino-6-(4-benzyloxy-3-chloro-phenylamino)-pyrimidine-5-carbaldehyde O-(2-morpholin-4-yl-ethyl)-oxime; MAPK, mitogen activated protein kinase; PLC, phospholipase C; PBS, phosphate-buffered salineAKT, protein kinase B; TGI, tumor growth inhibition; TTE, time to endpoint; ELISA, enzyme linked immunosorbent assay; ERK, extracellular signalregulated kinase; CDK, cyclin dependent kinase; BBB, blood-brain barrier.
Angiogenesis is a complex process that relies on a variety of growth factors and signaling pathways to stimulate endothelial cell responses and establish functional blood vessels. Signaling through the vascular endothelial growth factor (VEGF) receptors is an important mediator of angiogenesis, a hallmark of tumor growth and metastasis. Inhibition of signaling through VEGF has been clinically validated with FDA-approvals of bevacizumab, sorafenib, and suntinib. Our goal was to discover an orally available, selective VEGFR-2 inhibitor. A novel oxime, 1-{4-[6-amino-5-(methoxyimino-methyl)-pyrimidin-4-yloxy]-2-chloro-phenyl}-3-ethyl-urea (JNJ-38158471), was identified as a potent and selective inhibitor of VEGFR-2. While JNJ-38158471 shares some structure features with sorafenib, unlike sorafenib, it lacks Raf kinase activity. JNJ-38158471 inhibits VEGFR-2 (IC50 = 40 nM) and closely related tyrosine kinases, Ret (180 nM) and Kit (500 nM); it has no significant activity (>1 microM) against VEGFR-1 and VEGFR-3. At nanomolar levels, it inhibits VEGF-stimulated autophosphorylation of VEGFR-2 in a whole cell assay and inhibits VEGF-dependent endothelial migration. Once-daily oral dosing of JNJ-3815871 to nude mice bearing human A431, HCT116, and A375 tumors resulted in up to 90% tumor growth inhibition. Strikingly, after termination of JNJ-38158471 monotherapy-treatment of A375 xenografts, tumor growth delay was significantly prolonged up to 4 weeks. Anti-tumor efficacy correlated well with the observed dose concentrations (on a mg/kg basis) necessary to inhibit VEGF-induced corneal angiogenesis in C57BL/6J mice. In addition, the compound inhibited spontaneous polyp formation in the APC min-mouse model. These data demonstrate that JNJ-38158471 is a well tolerated, orally available, highly selective VEGFR-2 inhibitor that may have therapeutic benefit in human malignancies.
Erythropoietin (EPO) is an essential glycoprotein hormone that regulates erythrocyte production in hematopoietic tissues by stimulating growth, preventing apoptosis, and inducing the differentiation of red blood cell precursors. Clinically, EPO has been extensively used to increase levels of hemoglobin in the treatment of patients with anemia due to chronic renal failure, cancer chemotherapy, and HIV therapy. Recent studies suggest that the function of EPO and its cognate receptor, EPOR, are not strictly limited to erythroid lineages. EPOR expression has been identified in nonhematopoietic cells and tissues including endothelial, neuronal, and tumor cells. Epoetins have shown benefit in correcting anemia in chronic kidney disease and in chemotherapy-associated anemia in cancer patients. However, the incidence of thrombosis appears to be higher in patients administered EPO versus controls transfused to comparable hemoglobin levels. In particular, incidences seem higher in oncological patients treated with EPO and receiving combined radiation and chemotherapy than in those receiving chemotherapy alone. To address the potential interaction and responsiveness of the vasculature to EPO, we evaluated EPO using vascular endothelial cell assays. Biochemical analysis (eg, receptor expression, ligand-receptor binding, and activation of signaling cascades) as well as cellular analysis (eg, proliferation, migration, and differentiation) will be discussed. To evaluate the effects of EPO on thrombosis we used the in vivo pulmonary micoemboli (ME) model. In this fibrinolysis model, a preformed 125I-fibrin microemboli with a defined particle size is infused into mice intravenously. Microemboli lodge primarily in the lungs and the rate of radioactivity lost is measured over time. Defects in fibrinolysis have been identified in mice lacking either tPA or uPA. Mice were exposed acutely or chronically to EPO and the dissolution of ME was tested. Mice were injected subcutaneously with either a single dose of EPO (2500 IU/kg) and for the chronic studies EPO (1200 IU/kg) or control buffer three times a week for three weeks. The residual radioactivity in the lungs was measured after 10 and 90 minutes. Red blood cell count, hemoglobin, and hematocrit were analyzed weekly to confirm EPO activity. Our results indicate that neither a single (acute) dose of EPO nor chronic treatment using EPO affected the rate of pulmonary fibrinolysis. This suggests that EPO treatment does not significantly impair the function of the pulmonary fibrinolytic system proteins (eg, uPA, tPA, plasminogen, and PAI-1) using the ME model. We cannot exclude EPO’s potential influence on other aspects of coagulation and fibrinolysis.
Recombinant epoetins (epoetin alfa, darbepoetin alfa, epoetin beta) increase hemoglobin (Hb), reduce transfusions, and improve quality of life in patients with chemotherapy-related anemia. However, results from 2 phase III trials reporting lower survival rates relative to placebo in cancer patients treated with recombinant epoetins beyond anemia correction (ie, to Hb >12 g/dL) (Leyland-Jones. Lancet Oncol 2003; Henke et al. Lancet 2003) have prompted further investigation into the potential proliferative action of these agents on non-erythroid cells, including tumor cells, which express EPOR. The current analyses evaluated the clinical significance of EPOR expression after in vitro administration of recombinant human erythropoietin (rHuEPO, epoetin alfa), as well as the in vivo effect of recombinant epoetins on tumor growth, in 2 well-established preclinical models of breast carcinoma (MDA-MB-231 and MCF-7 cell lines). The in vitro analysis evaluated EPOR expression under hypoxic and normoxic conditions by immunoblotting, flow cytometry, and immunohistochemistry. EPOR binding was assessed with radioactive iodine-labeled rHuEPO (125I-rHuEPO). The in vivo analysis evaluated the effect of recombinant epoetin therapy on tumor growth in orthotopically implanted MDA-MB-231 and MCF-7 breast carcinoma xenograft models in athymic mice. Mice received 1 of 4 treatments: saline (control) every other day (QOD), 0.0025 mg/kg epoetin alfa subcutaneously (SC) QOD, 0.0075 mg/kg darbepoetin alfa SC once weekly, and 0.0025 mg/kg epoetin beta SC QOD. Effect on tumor growth was measured by calculating the difference in the final (Day 23) mean tumor volume between the treated group and the control group. Both cell lines demonstrated EPOR staining almost exclusively in the cytosol, with minimal cell surface expression. Intracellular EPOR was comparable under normoxic and hypoxic conditions, and hypoxia did not affect the expression or localization of EPOR. Epoetin alfa did not stimulate the migration, proliferation, or activation of signal transduction cascades in the 2 breast cancer models, although these pathways are normally activated in hematopoietic cells. There was no significant effect on tumor volume after 23 days of recombinant epoetin therapy compared with control. Mean tumor volumes ± standard error (SE) in the MDA-MB-231 cells on Day 23 were as follows: control, 847.6 ± 91.9 mm3; epoetin alfa, 560.6 ± 57.4 mm3; darbepoetin alfa, 809.8 ± 129.9 mm3; epoetin beta, 730.7 ± 66.4 mm3. In the MCF-7 cells, mean tumor volumes ± SE, respectively, were 1004.3 ± 72.9 mm3, 914.5 ± 245 mm3, 884.5 ± 97.1 mm3, and 809.4 ± 103.3 mm3. Recombinant epoetin therapy did not affect tumor inhibition or survival when coadministered with paclitaxel. In both cell lines, recombinant epoetin therapy resulted in mean final Hb values that were significantly (P<.01) higher than those observed with control (all agents produced Hb increases >1.0 g/dL/week), validating that pharmacologic doses were administered. Our findings suggest that although EPOR was expressed, it was nonfunctional and not involved in tumor growth promotion in these 2 models of breast carcinoma.
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