Osteosarcoma (OS) is the most common primary bone tumor in children and adolescents. Ninety percent of patients who present with metastatic and 30% to 40% of patients with nonmetastatic disease experience relapse, creating an urgent need for novel therapeutic strategies. The Met receptor tyrosine kinase and its ligand, hepatocyte growth factor (HGF), are important for mitosis, motility, and cell survival. Upregulation of Met/HGF signaling via receptor overexpression, amplification, or mutation drives the proliferation, invasiveness, and metastasis of a variety of cancer cells, including OS, prompting the development of Met/HGF inhibitors. OS cells depend on Met overexpression because introduction of dominant-negative Met inhibits in vivo tumorigenicity. Despite the importance of Met/HGF signaling in the development and maintenance of OS, the potential efficacy of pharmacologic Met inhibition in OS has been addressed only in in vitro studies. PF-2341066 is an orally bioavailable, selective ATP-competitive Met inhibitor that showed promising results recently in a phase I clinical trial in non-small cell lung cancer (NSCLC) patients. We tested the ability of PF-2341066 to inhibit malignant properties of osteosarcoma cells in vitro and orthotopic xenograft growth in vivo. In vitro, PF-2341066 inhibited osteosarcoma behavior associated with primary tumor growth (eg, proliferation and survival) as well as metastasis (eg, invasion and clonogenicity). In nude mice treated with PF-2341066 via oral gavage, the growth and associated osteolysis and extracortical bone matrix formation of osteosarcoma xenografts were inhibited by PF-2341066. PF-2341066 may represent an effective new systemic therapy for localized and potentially disseminated osteosarcoma. ß
These observations suggest a statistically significant reduction in serum PSA level that is associated with the onset of statin therapy.
Background Myocardial protection by anesthetics is known to involve activation of protein kinase C epsilon (PKCε). A key step in the activation process is auto-phosphorylation of the enzyme at serine 729. Our objectives were to identify the extent to which propofol interacts with PKCε and to identify the molecular mechanism(s) of interaction. Methods Immuno-blot analysis of recombinant PKCε was used to assess auto-phosphorylation of PKCε at serine 729 before and after exposure to propofol. An enzyme-linked immuno-absorbant assay kit was used for measuring PKC activity. Spectral shifts in fluorescence emission maxima of the C1B subdomain of PKCε in combination with the fluorescent phorbol ester, sapintoxin D, was used to identify molecular interactions between propofol and the phorbol ester/diacylglycerol binding site on the enzyme. Results Propofol (1 μM) caused a 6-fold increase in immuno-detectable, serine 729 phosphorylated PKCε and increased catalytic activity of the enzyme in a dose-dependent manner. DOG- or phorbol myristic acetate-induced activation of recombinant PKCε activity was enhanced by preincubation with propofol. Both propofol and phorbol myristic acetate quenched the intrinsic fluorescence spectra of the PKCε C1B subdomain in a dose-dependent manner, and propofol caused a further leftward-shift in the fluorescence emission maxima of sapintoxin D following addition of the C1B subdomain. Conclusions These results demonstrate that propofol interacts with recombinant PKCε causing auto-phosphorylation and activation of the enzyme. Moreover, propofol enhances phorbol ester-induced catalytic activity suggesting propofol binds to a region near the phorbol ester binding site allowing for allosteric modulation of PKCε catalytic activity.
Metastasis accounts for a majority of deaths from cancer. Metastatic melanoma is a type of highly aggressive cancer and accounts for ~75% of deaths in patients with skin cancer. The low 5-year survival rate of late-stage melanoma patients (~15%) treated with current methods calls for novel interventions. A constitutively active form of BRAF ( BRAFCA) is present in 60% to 70% of melanoma cases; the remaining 30% express wild type BRAF. Recently, a BRAFCA inhibitor, vemurafenib, has provided some initial benefits in melanoma patients, but resistance quickly developed in these patients. Also, the drug was ineffective in melanomas expressing wild type BRAF, begging for alternative therapeutic strategies. Metastasis is a multistep process, which involves detachment of cancer cells from primary tumor, intravasation into circulation, survival and extravasation into tissue parenchyma, and metastatic growth initiation and expansion. The last two steps are considered rate-limiting steps during metastasis and were examined using the experimental metastasis assay. Our prior studies showed that highly metastatic melanoma cells differentially express a set of 150 genes compared to low metastatic melanoma cells. Among the 150 genes, 44 were positively correlated with poor patient survival and were particularly enriched for genes encoding secreted proteins. We analyzed two of them, FZD7 and ERBB3, in the context of metastatic growth. FRIZZLED7 is a WNT receptor and ERBB3 is a receptor tyrosine kinase. Both were found to be highly expressed in melanomas from patients with poor survival. We showed that shRNA mediated knockdown of FZD7 or ERBB3 reduced the lung colonization ability of melanoma cell lines expressing the constitutively active mutant of BRAF (BRAFCA) in the experimental metastasis assay. In addition, FZD7 or ERBB3 knockdown reduced the metastatic potential of melanoma cells that have developed resistance to BRAF inhibitor. These results provided preclinical evidence for using FZD7 and ERBB3 as intervention entry points to treat both vemurafenib-sensitive and resistant melanomas. We are currently testing whether perturbing these two genes also abolishes metastasis from melanoma cells that carry wild type BRAF in the SKMEL2 cell line, which expresses a wild type BRAF. We are also examining at which step FZD7 and ERBB3 affect metastasis. Our data indicated that, though either FZD7 or ERBB3 knockdown can reduce the number of lung metastases in experimental metastasis assay, only FZD7 knockdown affected the tumor initiating cell frequency and subcutaneous tumor growth. This suggested that ERBB3 and FZD7 may regulate distinct aspects of metastasis. Taken together, we investigated the roles of two cell surface receptors, FZD7 and ERBB3, on melanoma metastasis. Our results revealed signaling pathways that potentially influence distinct steps of metastasis process. Combinatorial therapies that perturb these pathways thus may prove more effective in treating metastatic melanoma than single regimens. Citation Format: Shweta Tiwary, Morgan Preziosi, Sonali Mohanti, Brad Martin, Nathalie Zeitouni, Lei Xu. FRIZZLED7 and ERBB3 regulate distinct metastatic properties in melanoma. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C60.
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