Labeled EVs were added to 300 cells)

Labeled EVs were added to 300 cells). ErbB2; clathrin-independent internalization; and recycling. Our work suggests that, unlike Ras transformed cells, merlin-deficient cells do not depend on macropinocytic protein scavenging and instead exploit macropinocytosis for receptor recycling. Finally, we provide evidence that this macropinocytic proficiency of NF2-deficient cells can be used for therapeutic uptake. This work provides new insight into fundamental mechanisms of macropinocytic uptake and processing and suggests new ways to interfere with or exploit macropinocytosis in mutant and other tumors. expression or elimination of ezrin. Our data also suggest that deficiency in multiple tumor-relevant cell types, suggesting novel biomarkers and therapeutic insight into these intractable tumors. Results Merlin limits EGF-induced remodeling of cortical actomyosin Our previous studies revealed that merlin and ezrin influence the poorly comprehended interdependency between EGFR and the cortical cytoskeleton (Curto et al. 2007; Chiasson-MacKenzie et al. 2015). To better understand this relationship, we studied the cortical changes that occur in response to EGF stimulation in sections; and sections; 0.001, Mann-Whitney SCs. (**) 0.01; (***) 0.001, Mann-Whitney mutations underlie the development of schwannomas, meningiomas, and mesotheliomas in humans (Petrilli and Fernandez-Valle 2016). We found that EGF-induced ruffling and macropinocytosis were prominent in (Fig. 2D; Supplemental Fig. 2A). We then examined primary normal (confocal images showing CTxB-488 and N-WASP localization around the dorsal surface of EGF-stimulated 0.001, Mann-Whitney carrying a mutation in the ortholog exhibit excessive macropinocytosis (Bloomfield et al. 2015). Familial NF1, like NF2, features the development of SC-derived tumors known as neurofibromas but has distinct features or anatomical distribution relative to mutant schwannomas (Lin and Gutmann 2013). We found that, in contrast to control or 0.01; (***) 0.001, Mann-Whitney or Student’s 0.01; (***) 0.001, Mann-Whitney deficiency were also evident in mesothelioma cells (Supplemental Fig. 6E). Open in a separate window Physique 6. Merlin controls the surface levels of ErbB2 and spatial distribution of pAkt. (confocal images showing pAkt and F-actin in CDRs in EGF-stimulated 0.001, Mann-Whitney mutant tumors. First, a prediction of our studies is usually that NF2-deficient cells are addicted to the advantage conferred by the recycling of ErbB and perhaps other mitogenic receptors. Our results suggest that altered trafficking of EGFR in deficiency conferred increased sensitivity to ketoconazole, an inhibitor of sterol biosynthesis upstream of hydroxymethylsterol production (Fig. 7A). Importantly, this sensitivity was associated with a reduction in both Rab7 vesicle size and EGFR recycling (Fig. 7B,C). Thus, 0.01; (***) 0.001, Student’s and Mann-Whitney deficiency renders cells preferentially able to internalize fluorescently labeled EVs (PKH26-EVs). As shown in Physique 7D, deficiency on multiple signaling pathways. gene mutations occur in a range of human tumors (Petrilli and Fernandez-Valle 2016). Our work suggests new and broad ways to target mutant tumors. First, it may be possible to exploit their macropinocytic proficiency as a vulnerability. Unlike Ras transformed pancreatic tumor cells, mutant tumors may be sensitive to drugs such as ketoconazole that interfere with specific aspects of cholesterol homeostasis Daidzein or receptor recycling. These data also suggest that lysosomal protein degradation is not the only advantage that can be conferred by macropinocytosis. Second, mounting evidence suggests that macropinocytosis is an important route of therapeutic entry into cells (Ha et al. 2016). In proof-of-concept experiments we showed that Daidzein mutant and other tumor types. Materials and methods Cell culture LDCs are HB-like cells derived from hyperplastic liver lesions in a 12-wk-old mouse as described (Chiasson-MacKenzie et al. 2015). Cells were cultured in DMEM with 10% fetal bovine serum (FBS). Adenovirus contamination was used for re-expression (Ad5-CMV-or was deleted from SCs via adenovirus contamination with Cre-recombinase (Ad5-CMV-Cre). Human mesothelioma cell line HP-1 (provided by David Kwiatkowski, Brigham and Women’s Hospital, Boston) was produced in 10% FBS-DMEM. H2731 and NCI-H28 (provided by Cyril Benes, Massachusetts General Hospital Rabbit Polyclonal to DOK5 Center for Molecular Therapeutics, Boston) were cultured in 10% FBS-DMEM and 10% FBS-RPMI, respectively. expression construct was generated by PCR amplification of the mouse gene expression. An empty adenoviral vector was used as a control (EV). shRNA-expressing lentiviruses were generated by cotransfecting 293T cells with pLKO-puro.1 vectors and the packaging vectors VPR and VSVG (Fugene). Viruses were harvested 24C48 h after transfection. shScr- or shEzrin-expressing lentiviruses were stably expressed in LDCs and selected in 4 g/mL puromycin. Antibodies The following primary antibodies were used: anti-ezrin mouse monoclonal antibody (mAb; 1:500 for immunofluorescence; Neomarkers), anti-N-WASP rabbit polyclonal antibody (pAb; 1:100; Cell Signaling Technology),.2010). ezrin, altered actomyosin, and stabilized cholesterol-rich membranes. These changes profoundly alter EGF receptor (EGFR) trafficking in merlin-deficient cells, favoring increased membrane levels of its heterodimerization partner, ErbB2; clathrin-independent internalization; and recycling. Our work suggests that, unlike Ras transformed cells, merlin-deficient cells do not depend on macropinocytic protein scavenging and instead exploit macropinocytosis for receptor recycling. Finally, we provide evidence that this macropinocytic proficiency of NF2-deficient cells can be used for therapeutic uptake. This work provides new insight into fundamental mechanisms of macropinocytic uptake and processing and suggests new ways to interfere with or exploit macropinocytosis in mutant and other tumors. expression or elimination of ezrin. Our data also suggest that deficiency in multiple tumor-relevant cell types, suggesting novel biomarkers and therapeutic insight into these intractable tumors. Results Merlin limits EGF-induced remodeling of cortical actomyosin Our previous studies revealed that merlin and ezrin influence the poorly comprehended interdependency between EGFR and the cortical cytoskeleton (Curto et al. 2007; Chiasson-MacKenzie et al. 2015). To better understand this relationship, we studied the cortical changes that occur in response to EGF stimulation in sections; and sections; 0.001, Mann-Whitney SCs. (**) 0.01; (***) 0.001, Mann-Whitney mutations underlie the development of schwannomas, meningiomas, and mesotheliomas in humans (Petrilli and Fernandez-Valle 2016). We found that EGF-induced ruffling and macropinocytosis were prominent in (Fig. 2D; Supplemental Fig. 2A). We then examined primary normal (confocal images showing CTxB-488 and N-WASP localization around the dorsal surface of EGF-stimulated 0.001, Mann-Whitney carrying a mutation in the ortholog exhibit excessive macropinocytosis (Bloomfield et al. 2015). Familial NF1, like NF2, features the development of SC-derived tumors known as neurofibromas but has distinct features or anatomical distribution relative to mutant schwannomas (Lin and Gutmann 2013). We found that, in contrast to control or 0.01; (***) 0.001, Mann-Whitney or Student’s 0.01; (***) 0.001, Mann-Whitney deficiency were also evident in mesothelioma cells (Supplemental Fig. 6E). Open in a separate window Physique 6. Merlin controls the surface levels of ErbB2 and spatial distribution of pAkt. (confocal images showing pAkt and F-actin in CDRs in EGF-stimulated 0.001, Mann-Whitney mutant tumors. First, a prediction of our studies is usually that NF2-deficient cells are addicted to the advantage conferred by the recycling of ErbB and perhaps other mitogenic receptors. Our results suggest that altered trafficking of EGFR in deficiency conferred increased sensitivity to ketoconazole, an inhibitor of sterol biosynthesis upstream of hydroxymethylsterol production (Fig. 7A). Importantly, this sensitivity was associated with a reduction in both Rab7 vesicle size and EGFR recycling (Fig. 7B,C). Thus, 0.01; (***) 0.001, Student’s and Mann-Whitney deficiency renders cells preferentially able to internalize fluorescently labeled EVs (PKH26-EVs). As shown in Figure 7D, deficiency on multiple signaling pathways. gene mutations occur in a range of human tumors (Petrilli and Fernandez-Valle 2016). Our work suggests new and broad ways to target mutant tumors. First, it may be possible to exploit their macropinocytic proficiency as a vulnerability. Unlike Ras transformed pancreatic tumor cells, mutant tumors may be sensitive to drugs such as ketoconazole that interfere with specific aspects of cholesterol homeostasis or receptor recycling. These data also suggest that lysosomal protein degradation is not the only advantage that can be conferred by macropinocytosis. Second, mounting evidence suggests that macropinocytosis is an important route of therapeutic entry into cells (Ha et al. 2016). In proof-of-concept experiments we showed that mutant and other tumor types. Materials and methods Cell culture LDCs are HB-like cells derived from hyperplastic liver lesions in a 12-wk-old mouse as described (Chiasson-MacKenzie et al. 2015). Cells were cultured in DMEM with 10% fetal bovine serum (FBS). Adenovirus infection was used for re-expression (Ad5-CMV-or was deleted from SCs via adenovirus infection with Cre-recombinase (Ad5-CMV-Cre). Human mesothelioma cell line HP-1 (provided by David Kwiatkowski, Brigham and Women’s Hospital, Boston) was grown in 10% FBS-DMEM. H2731 and NCI-H28 (provided by Cyril Benes, Massachusetts General Hospital Center for Daidzein Molecular Therapeutics, Boston) were cultured in 10% FBS-DMEM and 10% FBS-RPMI, respectively. expression construct was generated by PCR amplification of the mouse gene expression. An empty adenoviral vector was used as a control (EV)..