Background Mast cell activation is an important driver of abdominal pain in irritable bowel syndrome (IBS). While evidence supports the role of IgE-mediated mast cell activation in visceral pain development in IBS, the role of pseudoallergic MRGPRX2-mediated mast cell activation in this process remains unknown. Objective We investigated whether MRGPRX2-mediated mast cell activation plays a role in abdominal pain development in patients with IBS. Design MRGPRX2 expression in mast cells and other immune cells was characterised across colon layers using flow cytometry. We evaluated whether MRGPRX2 agonists trigger mast cell degranulation and transient receptor potential vanilloid 1 (TRPV1) sensitisation in healthy human colonic submucosal plexus samples using live imaging. Rectal biopsies were then collected from patients with IBS and healthy volunteers (HV) and MRGPRX2 + mast cell frequency, MRGPRX2 expression per cell, mast cell degranulation kinetics in response to MRGPRX2 agonists, MRGPRX2 agonistic activity and presence of MRGPRX2 agonists in biopsy supernatants were assessed. Results MRGPRX2 + mast cells are enriched in the submucosa and muscularis of the healthy human colon. MRGPRX2 agonists induce mast cell degranulation and TRPV1 sensitisation in the healthy colon submucosa. While the frequency of rectal MRGPRX2 + mast cells was unaltered in IBS, submucosal mast cells showed increased degranulation in response to MRGPRX2 agonists in IBS compared with HV. MRGPRX2 agonistic activity was increased in IBS rectal biopsy supernatant compared with HV, which was associated with increased levels of substance P. Conclusion The MRGPRX2 pathway is functionally upregulated in the colon of patients with IBS, supporting its role in abdominal pain in IBS.
Glioblastoma is both the most common and lethal primary malignant brain tumor. Extensive multiplatform genomic characterization has provided a higher-resolution picture of the molecular alterations underlying this disease. These studies provide the emerging view that "glioblastoma" represents several histologically similar yet molecularly heterogeneous diseases, which influences taxonomic classification systems, prognosis, and therapeutic decisions.
Abstract EGFR-targeted therapies such as the tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib have had limited success clinically in glioblastoma (GBM). However, some of the most common mechanisms of resistance to these agents in other solid tumors are rarely present in GBM. In an effort to identify molecular mechanisms of acquired resistance to EGFR TKIs in GBM, we utilized ink4a/arf −/− astrocytes over-expressing EGFR and having wild-type or deleted PTEN. To model TKI-resistance in vitro, cells were seeded in soft agar with 1 μM gefitinib or erlotinib for 2 weeks; during this time the growth of colonies was inhibited. The dose was escalated to 2 μM and after 3 weeks, TKI-resistant colonies began to form and were isolated to generate clonal cell lines. In three PTEN wild-type TKI-resistant lines, EGFR expression was not altered and gefitinib inhibited receptor phosphorylation in a manner identical to that observed in parental cells, thereby ruling out the possibility that the cells are resistant because the drug cannot reach its site of action and inhibit its target. Interestingly, two of these three cell lines demonstrated decreased PTEN expression and increased AKT phosphorylation. The remaining cell line had no change in total PTEN levels, and appeared identical to parental cells with regard to AKT phosphorylation. Removal of resistant cells from the maintenance dose of TKI resulted in restoration of EGFR and ERK phosphorylation to a level several-fold greater than that in parental cells. Notably, the TKI-resistant phenotype was reversible, and these cells became significantly more sensitive to gefitinib and erlotinib than parental cells. Overall, we have generated and characterized a model system for the identification of mechanisms of resistance to EGFR TKIs in GBM. We have identified at least two different resistance scenarios in cells with wild-type PTEN: decrease in PTEN expression, and another as-of-yet unknown mechanism that does not appear to involve changes in the PI3K pathway. The reversibility of resistance and subsequent increased sensitivity to TKIs have potential clinical implications. This work forms the basis for our effort to uncover novel targets or approaches to therapy that may increase the utility of these drugs for treatment of GBM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B73.
Circulating monocytes (Mo) are precursors to a subset of gastric resident-muscularis macrophages. Changes in muscularis-macrophages (MMs) result in delayed gastric emptying (DGE) in diabetic gastroparesis. However, the dynamics of Mo in the development of DGE in an animal model are unknown. Using CyTOF and computational approaches, we show a high heterogeneity within the Mo-population. In DGE mice, via unbiased clustering, we identified two reduced Mo clusters which exhibit migratory phenotype (Ly6C hi CCR2 hi-int CD62L hi Ly6G hi CD45R hi MERTK hiint LGALS3 int CD14 int CX3CR1 low Siglec-H int-low ) resembling classical-Mo (CMo-like). All markers enriched in these clusters are known to regulate cell differentiation, proliferation, adhesion, and migration. Trajectory inference analysis predicted these Mo as precursors to subsequent Mo-lineages. In gastric muscle tissue, we demonstrated an increase in the gene expression levels of chemokine receptor Ccr2 and its ligand Ccl2, suggesting increased trafficking of classical-Mo. These findings establish a link between two CMo-like clusters and the development of DGE phenotype and contribute to better understanding of the heterogenicity of the Mo-population.
Receptor tyrosine kinases (RTKs) such as the epidermal growth factor receptor (EGFR) regulate cellular homeostatic processes. EGFR activates downstream signaling cascades that promote tumor cell survival, proliferation and migration. Dysregulation of EGFR signaling as a consequence of overexpression, amplification and mutation of the EGFR gene occurs frequently in several types of cancers and many become dependent on EGFR signaling to maintain their malignant phenotypes. Consequently, concerted efforts have been mounted to develop therapeutic agents and strategies to effectively inhibit EGFR. However, limited therapeutic benefits to cancer patients have been derived from EGFR-targeted therapies. A well-documented obstacle to improved patient survival is the presence of EGFR-inhibitor resistant tumor cell variants within heterogeneous tumor cell masses. Here, we summarize the mechanisms by which tumors resist EGFR-targeted therapies and highlight the emerging role of microRNAs (miRs) as downstream effector molecules utilized by EGFR to promote tumor initiation, progression and that play a role in resistance to EGFR inhibitors. We also examine evidence supporting the utility of miRs as predictors of response to targeted therapies and novel therapeutic agents to circumvent EGFR-inhibitor resistance mechanisms.
EphrinA1 is a glycosylphosphatidylinositol (GPI)-linked ligand for the EphA2 receptor, which is overexpressed in glioblastoma (GBM), among other cancers. Activation of the receptor by ephrinA1 leads to a suppression of oncogenic properties of GBM cells. We documented that a monomeric functional form of ephrinA1 is released from cancer cells and thus explored the mechanism of ephrinA1 release and the primary protein sequence. We demonstrate here that multiple metalloproteases (MMPs) are able to cleave ephrinA1, most notably MMP-1, -2, -9, and -13. The proteolytic cleavage that releases ephrinA1 occurs at three positions near the C terminus, producing three forms ending in valine-175, histidine-177, or serine-178. Moreover, deletion of amino acids 174 to 181 or 175 to 181 yields ephrinA1 that is still GPI linked but not released by proteolysis, underlining the necessity of amino acids 175 to 181 for release from the membrane. Furthermore, recombinant ephrinA1 ending at residue 175 retains activity toward the EphA2 receptor. These findings suggest a mechanism of release and provide evidence for the existence of several forms of monomeric ephrinA1. Moreover, ephrinA1 should be truncated at a minimum at amino acid 175 in fusions or conjugates with other molecules in order to prevent likely proteolysis within physiological and pathobiological environments.
Glioblastoma multiforme (GBM) is the most aggressive of the astrocytic malignancies and the most common intracranial tumor in adults. Although the epidermal growth factor receptor (EGFR) is overexpressed and/or mutated in at least 50% of GBM cases and is required for tumor maintenance in animal models, EGFR inhibitors have thus far failed to deliver significant responses in GBM patients. One inherent resistance mechanism in GBM is the coactivation of multiple receptor tyrosine kinases, which generates redundancy in activation of phosphoinositide-3'-kinase (PI3K) signaling. Here we demonstrate that the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumor suppressor is frequently phosphorylated at a conserved tyrosine residue, Y240, in GBM clinical samples. Phosphorylation of Y240 is associated with shortened overall survival and resistance to EGFR inhibitor therapy in GBM patients and plays an active role in mediating resistance to EGFR inhibition in vitro. Y240 phosphorylation can be mediated by both fibroblast growth factor receptors and SRC family kinases (SFKs) but does not affect the ability of PTEN to antagonize PI3K signaling. These findings show that, in addition to genetic loss and mutation of PTEN, its modulation by tyrosine phosphorylation has important implications for the development and treatment of GBM.
