Obesity-Induced Colorectal Cancer Is Driven by Caloric Silencing of the Guanylin-GUCY2C Paracrine Signaling Axis.

Obesity is a well-known risk factor for colorectal cancer but precisely how it influences risks of malignancy remains unclear. During colon cancer development in humans or animals, attenuation of the colonic cell surface receptor guanylyl cyclase C (GUCY2C) that occurs due to loss of its paracrine hormone ligand guanylin contributes universally to malignant progression. In this study, we explored a link between obesity and GUCY2C silencing in colorectal cancer. Using genetically engineered mice on different diets, we found that diet-induced obesity caused a loss of guanylin expression in the colon with subsequent GUCY2C silencing, epithelial dysfunction, and tumorigenesis. Mechanistic investigations revealed that obesity reversibly silenced guanylin expression through calorie-dependent induction of endoplasmic reticulum stress and the unfolded protein response in intestinal epithelial cells. In transgenic mice, enforcing specific expression of guanylin in intestinal epithelial cells restored GUCY2C signaling, eliminating intestinal tumors associated with a high calorie diet. Our findings show how caloric suppression of the guanylin-GUCY2C signaling axis links obesity to negation of a universal tumor suppressor pathway in colorectal cancer, suggesting an opportunity to prevent colorectal cancer in obese patients through hormone replacement with the FDA-approved oral GUCY2C ligand linaclotide

Abstracts from the 8th International Conference on cGMP Generators, Effectors and Therapeutic Implications

This work was supported by a restricted research grant of Bayer AG

Intra- and inter-molecular interactions regulate the activity of the Rac-GAPs α2- and β2-chimaerins

Chimaerins are a family of GTPase activating proteins (GAPs) for the small G-protein Rac that have gain recent attention due to their important roles in development, cancer, neuritogenesis, and T-cell function. Chimaerins possess a C1 domain capable of binding phorbol esters and the lipid second messenger diacylglycerol (DAG) in vitro. Here we identified the N- and C-termini of α2-chimaerin as autoinhibitory domains that restrict ligand binding to the C1 domain and activation of its Rac-GAP activity. Using biochemical and modeling analysis we identified residues that are involved in intramolecular interactions and are key to stabilize the protein in its inactive state. Mutation of these sites renders α2-chimaerin hypersensitive to C1 ligands, as reflected by its enhanced ability to translocate in response to PMA and to inhibit Rac activity and cell migration. Furthermore, we demonstrate for the first time that α2-chimaerin is an effector of the EGFR. EGF treatment induces α2-chimaerin translocation to the plasma membrane in a DAG-dependent manner. A C1 domain mutant with reduced affinity for DAG is unable to translocate in response to EGF. α2-chimaerin expression in HeLa cells blocks EGF-induced Rac activation, indicating that EGF-mediated activation of α2-chimaerin serves to self-limit Rac activation. In addition, we present evidence showing that α2- and β2-chimaerins bind to the adaptor protein Nck1. Deletional and mutational analysis allowed us to identify a novel non-typical proline rich region conserved in α2- and β2-chimaerin that interacts with the third SH3 domain of Nck1. Treatment with PMA at concentrations that translocate chimaerins induces the dissociation of the Nck-chimaerin complex. Moreover, preliminary data indicates that overexpression of Nck1 inhibits PMA-induced translocation of α2-chimaerin. Modeling analysis of β2-chimaerin in complex with the third SH3 domain of Nck suggests that Nck binding is specific to the inactive conformation. This was supported by the observation that hyperactivated mutants of α2- and β2-chimaerin show decreased binding to Nck. Based on our results we propose, that the activation of α2- and β2-chimaerins is regulated both by intra- and inter-molecular interactions and by DAG binding to the C1 domain

Intra- and inter-molecular interactions regulate the activity of the Rac-GAPs α2- and β2-chimaerins

Chimaerins are a family of GTPase activating proteins (GAPs) for the small G-protein Rac that have gain recent attention due to their important roles in development, cancer, neuritogenesis, and T-cell function. Chimaerins possess a C1 domain capable of binding phorbol esters and the lipid second messenger diacylglycerol (DAG) in vitro. Here we identified the N- and C-termini of α2-chimaerin as autoinhibitory domains that restrict ligand binding to the C1 domain and activation of its Rac-GAP activity. Using biochemical and modeling analysis we identified residues that are involved in intramolecular interactions and are key to stabilize the protein in its inactive state. Mutation of these sites renders α2-chimaerin hypersensitive to C1 ligands, as reflected by its enhanced ability to translocate in response to PMA and to inhibit Rac activity and cell migration. Furthermore, we demonstrate for the first time that α2-chimaerin is an effector of the EGFR. EGF treatment induces α2-chimaerin translocation to the plasma membrane in a DAG-dependent manner. A C1 domain mutant with reduced affinity for DAG is unable to translocate in response to EGF. α2-chimaerin expression in HeLa cells blocks EGF-induced Rac activation, indicating that EGF-mediated activation of α2-chimaerin serves to self-limit Rac activation. In addition, we present evidence showing that α2- and β2-chimaerins bind to the adaptor protein Nck1. Deletional and mutational analysis allowed us to identify a novel non-typical proline rich region conserved in α2- and β2-chimaerin that interacts with the third SH3 domain of Nck1. Treatment with PMA at concentrations that translocate chimaerins induces the dissociation of the Nck-chimaerin complex. Moreover, preliminary data indicates that overexpression of Nck1 inhibits PMA-induced translocation of α2-chimaerin. Modeling analysis of β2-chimaerin in complex with the third SH3 domain of Nck suggests that Nck binding is specific to the inactive conformation. This was supported by the observation that hyperactivated mutants of α2- and β2-chimaerin show decreased binding to Nck. Based on our results we propose, that the activation of α2- and β2-chimaerins is regulated both by intra- and inter-molecular interactions and by DAG binding to the C1 domain

Evaluation of a Cerebral-Blood-Volume (CBV) pharmaco-MRI (phMRI) assay utilizing low (0.1mg/70kg) and high (0.2mg/70kg) dose buprenorphine infusion and a novel USPIO contrast agent (Ferumoxytol) in healthy human subjects

We present results from a clinical trial of pharmaco-MRI (phMRI) employing cerebral blood volume (CBV) imaging using ferumoxytol (Rienso/Feraheme, AMAG) as a blood pool contrast agent. The study examined the pharmacodynamic effects of two single doses of buprenorphine (0.2mg/70kg and 0.1mg/70kg administered intravenously). We found that contrast-enhanced CBV phMRI signals are more sensitive reporters of pharmacodynamic effects than conventional blood oxygen level dependent (BOLD) phMRI. In particular, higher sensitivity of CBV phMRI compared to BOLD allows for elucidation of PD responses at lower doses of buprenorphine, which has practical implications for similar phMRI studies with centrally acting drugs