Biomedical subjects
Publications and source records attributed to Scott A Boerner.
Finding new therapies to assist in the treatment of cancer is a major challenge of clinical research. Small molecules that inhibit different molecular targets at the different levels of the MAPK pathway have been developed. Several MEK inhibitors have been examined in early-phase clinical trials and the current state of clinical results using these therapies is presented here.
Activation of the Ras-MAPK signal transduction pathway is necessary for biological responses both to growth factors and ECM. Here, we provide evidence that phosphorylation of S298 of MAPK kinase 1 (MEK1) by p21-activated kinase (PAK) is a site of convergence for integrin and growth factor signaling. We find that adhesion to fibronectin induces PAK1-dependent phosphorylation of MEK1 on S298 and that this phosphorylation is necessary for efficient activation of MEK1 and subsequent MAPK activation. The rapid and efficient activation of MEK and phosphorylation on S298 induced by cell adhesion to fibronectin is influenced by FAK and Src signaling and is paralleled by localization of phospho-S298 MEK1 and phospho-MAPK staining in peripheral membrane-proximal adhesion structures. We propose that FAK/Src-dependent, PAK1-mediated phosphorylation of MEK1 on S298 is central to the organization and localization of active Raf-MEK1-MAPK signaling complexes, and that formation of such complexes contributes to the adhesion dependence of growth factor signaling to MAPK.
Integrins are transmembrane proteins that serve as primary sensors of the extracellular matrix (ECM) environment. In response to interactions with the ECM, integrins initiate signaling pathways that regulate cell migration, growth, and survival. Advances in imaging have contributed to the understanding of the dynamic nature of these cell-ECM interactions and the complexes that form at these sites and have provided insights into their regulation and signal organizing functions.
Focal adhesion kinase (FAK) is a regulator of numerous adhesion-dependent processes including cell migration, cell proliferation, and cell survival. The C-terminal domain of FAK, FAK-related nonkinase (FRNK), is autonomously expressed and functions as an inhibitor of FAK signaling. Previous attempts to use FRNK as a tool to dissect FAK signaling have been limited because of an inability to temporally regulate the inhibitory functions of FRNK. In this report, we describe and characterize a conditionally targeted form of FRNK that was created by fusing the hormone-binding domain of the estrogen receptor (ER*) to the C-terminus of FRNK. In the absence of added hormone, FRNK-ER* was diffusely distributed throughout the cytoplasm of cells. Upon addition of hormone, the cytoplasmic pool of FRNK-ER* was rapidly redistributed to focal adhesions. We demonstrate that cells expressing FRNK-ER* show a hormone-dependent decrease in FAK tyrosine phosphorylation and cell migration. Furthermore, when cells expressing of FRNK-ER* were treated with hormone, the cells responded with a dramatic change in cell morphology, suggesting a role for FAK in the regulation of the adhesive properties of focal adhesions.
MCF-7 human breast cancer cells are widely utilized to study apoptotic processes. Recent studies demonstrated that these cells lack procaspase-3. In the present study, caspase activation and activity were examined in this cell line after treatment with the microtubule poison paclitaxel. When cells were harvested 72 h after the start of a 24-h treatment with 100 nm paclitaxel, 37 +/- 5% of the cells were nonadherent and displayed apoptotic morphological changes. Although mitochondrial cytochrome c release and caspase-9 cleavage were detectable by immunoblotting, assays of cytosol and nuclei prepared from the apoptotic cells failed to demonstrate the presence of activity that cleaved the synthetic caspase substrates LEHD-7-amino-4-trifluoromethylcoumarin (LEHD-AFC), DEVD-AFC, and VEID-AFC. Likewise, the paclitaxel-treated MCF-7 cells failed to cleave a variety of caspase substrates, including lamin A, beta-catenin, gelsolin, protein kinase Cdelta, topoisomerase I, and procaspases-6, -8, and -10. Transfection of MCF-7 cells with wild type procaspase-3 partially restored cleavage of these polypeptides but did not result in detectable activities that could cleave the synthetic caspase substrates. Immunoblotting revealed that caspase-9, and -3, which were proteolytically cleaved in paclitaxel-treated MCF-7/caspase-3 cells, were sequestered in a salt-resistant sedimentable fraction rather than released to the cytosol. Immunofluorescence indicated large cytoplasmic aggregates containing cleaved caspase-3 in these apoptotic cells. These observations suggest that sequestration of caspases can occur in some model systems, causing tetrapeptide-based activity assays to underestimate the amount of caspase activation that has occurred in situ.
The purpose of this investigation was to evaluate the occurrence of autoimmune toxicities associated with the administration of low-dose IL-2 in conjunction with vaccines for melanoma. Ninety-three patients with stage IIB, III, or IV melanoma were enrolled in three clinical trials and received anti-melanoma vaccines on days 1, 8, 15, 29, 36, and 43. The vaccines comprised peptide-pulsed dendritic cells, autologous tumor cells with GM-CSF in Montanide ISA-51, or synthetic peptides with GM-CSF in Montanide ISA-51. In conjunction with the vaccines, all patients were administered 3 x 10(6) IU/m2/d IL-2 subcutaneously for 42 days, either days 8 to 49 or 29 to 70. Clinical and laboratory data from these studies were reviewed in aggregate to evaluate the occurrence of autoimmune toxicities. Of 91 evaluable patients, vitiligo was documented in 6 patients (7%). In addition, one patient experienced transient severe insulin-dependent diabetes that resolved after discontinuing IL-2, and another experienced an exacerbation of his pre-existing diabetes; these occurrences are consistent with an autoimmune insulitis. Four occurrences (4%) of transient minor ocular toxicity were documented, but no autoimmune ocular toxicities or changes in visual acuity were found. Of 55 evaluable patients, 14 experienced thyroid abnormalities (25%). These were attributed to an autoimmune thyroiditis, which was supported by findings of antithyroid antibodies in three of the seven patients evaluated. Overall, autoimmune toxicities affecting several organ systems were observed in patients receiving melanoma vaccines in conjunction with low-dose IL-2. None of these toxicities caused major long-term effects, though one was acutely life-threatening and others contributed to treatment-related morbidity. Peptide- or cell-based vaccines administered in combination with low-dose IL-2 appear to be capable of breaking tolerance to self-antigens; despite the associated toxicities, these combinations may still be useful to administer as an immunotherapy for cancer. However, careful monitoring for autoimmune toxicities should be incorporated in future clinical studies incorporating low-dose IL-2.