Opioid drugs are widely used analgesics that activate the G protein-coupled µ-opioid receptor, whose endogenous neuropeptide agonists, endorphins and enkephalins, are potent pain relievers. The ...therapeutic utility of opioid drugs is hindered by development of tolerance to the analgesic effects, requiring dose escalation for persistent pain control and leading to overdose and fatal respiratory distress. The prevailing hypothesis is that the intended analgesic effects of opioid drugs are mediated by µ-opioid receptor signaling to G protein, while the side-effects of respiratory depression and analgesic tolerance are caused by engagement of the receptor with the arrestin-3 protein. Consequently, opioid drug development has focused exclusively on identifying agonists devoid of arrestin-3 engagement. Here, we challenge the prevailing hypothesis with a panel of six clinically relevant opioid drugs and mice of three distinct genotypes with varying abilities to promote morphine-mediated arrestin-3 engagement. With this genetic and pharmacological approach, we demonstrate that arrestin-3 recruitment does not impact respiratory depression, and effective arrestin-3 engagement reduces, rather than exacerbates, the development of analgesic tolerance. These studies suggest that future development of safer opioids should focus on identifying opioid ligands that recruit both G protein and arrestin-3, thereby mimicking the signaling profile of most endogenous µ-opioid receptor agonists.
Type 2 diabetes is characterized by disruption of glucose homeostasis. Insulin is the primary driver of glucose homeostasis and released by pancreatic β cells in response to nutritional and endocrine ...signals. Insulin release is also stimulated by incretin hormones including Glucagon-Like peptide 1 (GLP-1) which acts on the GLP-1 receptor (GLP1-R) in islet producing the “Incretin effect.” Common genetic variations in GLP-1R alter this incretin effect. Specifically, the R131Q variant increases while the G168S variant decreases the effect of GLP-1. We have found that these genetic variations do not affect the affinity of GLP-1 nor its efficacy for G protein signaling. Taken together, these findings present a “pharmacological mystery” as to how genetic variation at GLP-1R affects incretin function. In this study, we assessed whether genetic variation in GLP-1R alters receptor trafficking resulting in differential incretin response. HEK293 cells stably expressing wild type (WT), R131Q, and G168S variants were used to measure receptor endocytosis and post-endocytic sorting to the lysosome. We found that WT GLP-1R degrades after endocytosis via its interaction with the GPCR-associated sorting protein-1 (GASP1), a protein highly expressed in islets. Importantly, we found that GLP-1R variant R131Q recycled following endocytosis while variant G168S was more rapidly degraded compared to the WT GLP-1R. To assess whether GASP1 mediated GLP-1R degradation affects incretin function, we used CRISPR to generate rat INS-1 cells with a knockout (KO) of GASP1. Deletion of GASP1 had no effect on acute GLP-1R signaling. However, prolong agonist pretreatment produced a loss of the incretin response in WT INS-1 cells, while GASP1 KO cells showed preservation of the incretin effect. These results suggest GASP1 mediates the post-endocytic degradation of GLP-1R in β cells and that altered trafficking of the GLP-1R variants may underlie differential incretin response in humans who carry these variants.
Disclosure
A. Gaur: None.
Funding
University of California, Davis
CD133, known as prominin1, is a penta-span transmembrane glycoprotein presumably a cancer stem cell marker for carcinomas, glioblastomas, and melanomas. We showed that CD133(+) ‘melanoma-initiating ...cells’ are associated with chemoresistance, contributing to poor patient outcome. The current study investigates the role(s) of CD133 in invasion and metastasis. Magnetic-activated cell sorting of a melanoma cell line (BAKP) followed by transwell invasion assays revealed that CD133(+) cells are significantly more invasive than CD133(−) cells. Conditional reprogramming of BAKP CD133(+) cells maintained stable CD133 overexpression (BAK-R), and induced cancer stem cell markers, melanosphere formation, and chemoresistance to kinase inhibitors. BAK-R cells showed upregulated CD133 expression, and consequently were more invasive and metastatic than BAK-P cells in transwell and zebrafish assays. CD133 knockdown by siRNA or CRISPR-Cas9 (BAK-R-T3) in BAK-R cells reduced invasion and levels of matrix metalloproteinases MMP2/MMP9. BAK-R-SC cells, but not BAK-R-T3, were metastatic in zebrafish. While CD133 knockdown by siRNA or CRISPR-Cas9 in BAK-P cells attenuated invasion and diminished MMP2/MMP9 levels, doxycycline-induced CD133 expression in BAK-P cells enhanced invasion and MMP2/MMP9 concentrations. CD133 may therefore play an essential role in invasion and metastasis via upregulation of MMP2/MMP9, leading to tumor progression, and represents an attractive target for intervention in melanoma.
Wound healing requires well-coordinated events including hemostasis, inflammation, proliferation, and remodeling. Delays in any of these stages leads to chronic wounds, infections, and hypertrophic ...scarring. Burn wounds are particularly problematic, and may require intervention to ensure timely progression to reduce morbidity and mortality. To accelerate burn wound healing, Platelet-Rich Plasma (PRP)1 can be of value, since platelets release growth factor proteins and inorganic polyphosphates (polyP) that may be integral to wound healing. We used polyP-depleted keratinocyte (HaCaT) and fibroblast cell culture models to determine cell proliferation and scratch-wound repair to determine if polyP, platelet lysate, or combined treatment could accelerate wound healing. While polyP and PRP significantly reduced the open scratch-wound area in fibroblasts and keratinocytes, polyP had no effect on keratinocyte or fibroblast proliferation. PRP was also evaluated as a treatment in a murine model of full thickness wound healing in vivo, including a treatment in which PRP was supplemented with purified polyP. PRP induced significantly more rapid re-epithelialization by Day 3. Pure polyP enhanced the effects of PRP on epithelial tongues, which were significantly elongated in the PRP + high-dose polyP treatment groups compared to PRP alone. Thus, PRP and polyP may serve as an effective therapeutic combination for treating wounds.
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The increasing prevalence of type 2 diabetes (T2D) and obesity poses substantial concerns and economic challenges to the healthcare system. T2D is characterized by disruption of glucose homeostasis. ...Insulin is the primary driver of glucose homeostasis and released by pancreatic β-cells in response to nutritional and endocrine signals. Glucose stimulated insulin secretion is augmented by the actions of incretin hormones including Glucagon-Like peptide 1 (GLP-1), which activates the GLP-1 receptor (GLP-1R) in islets producing the “incretin effect”. Because of their insulinotropic properties, incretin drugs, such as GLP-1 receptor agonists are used as therapeutic agents to maintain glucose homeostasis in T2D. However, concerns regarding the long-term effects of these drugs and development of tolerance to GLP-1R agonist persist. In this study, we investigate the role of G protein-coupled receptor-associated sorting protein 1 (GASP1), a critical regulator involved in post-endocytic trafficking of GLP-1R, on development of tolerance to GLP-1R agonists. By combining CRISPR-Cas9 technique, Homogenous Time Resolved Fluorescence (HTRF) biochemical assay, transgenic mice and in vivo and ex vivo animal studies, we found the following:• GASP1 is expressed in both human embryonic kidney (HEK 293) and rat insulinoma derived insulin producing INS-1 cells.• Following acute treatment with the GLP-1R agonist Exendin-4 (Ex-4), both HEK 293 and INS-1 cells demonstrate a dose-dependent increase in intracellular cyclic adenosine monophosphate (cAMP) levels.• Prolonged Ex-4 pretreatment of HEK 293 and INS-1 cells with Ex-4 results in loss of responsiveness of the receptor (i.e “Tolerance”).• CRISPR-Cas9-mediated removal of GASP1 in both HEK 293 and INS-1 cells did not impact acute GLP-1R signaling. However, GASP1 knockout prevented the development of tolerance in response to prolonged Ex-4 pretreatment, indicating that GASP1 plays a role in regulating the post-endocytic trafficking of GLP-1R.• Similarly, in INS-1 cells, deletion of GASP1 has no effect on incretin-mediated insulin secretion in response to Ex-4. However, upon prolonged Ex-4 pretreatment wild-type (WT) INS-1 cells show reduced incretin response, whereas GASP1 knockout (KO) INS-1 cells retained the incretin effect.• Furthermore, in a longitudinal mouse islet insulin secretion assay, both GASP1 wild-type and beta-cell specific GASP1 deleted (β-GASP1-KO) islets show robust incretin effect and increase in insulin secretion when stimulated with Ex-4 acute treatment. • Interestingly, after an Ex-4 pretreatment, GASP1-WT islets show reduced insulin secretion indicating development of tolerance while β-GASP1-KO islets maintained their incretin effect.• Importantly, both GASP1-WT and β-GASP1-KO islets displayed a substantial incretin effect after a 24-hour recovery period, suggesting that the observed tolerance effect is not due to any inherent unhealthiness of the islets.• In WT mice which are chronically treated with Ex-4 for six weeks show development of tolerance to glucose-stimulated insulin secretion effect of Ex-4.• Furthermore, in WT mice treated chronically with Ex-4, there is not only the development of tolerance to exogenous Ex-4 treatment but also to their endogenous incretins.• In mice with selective disruption of GASP1 in pancreatic beta cells (β-GASP1-KO mice), the acute treatment of oral glucose and Ex-4 are indistinguishable from WT mice. • However, chronic Ex-4 treatment of β-GASP1-KO mice with Ex-4 does not develop tolerance to either exogenous Ex-4 or endogenous incretins.This study highlights the pivotal role of GASP1 in regulating the post-endocytic trafficking of GLP-1R in pancreatic β-cells and its impact on receptor function during prolonged drug administration. These findings also emphasize the critical role of GASP1-mediated GLP-1R trafficking in the development of tolerance to incretin drugs and offers potential novel strategies for improving therapeutic efficacy. Therefore, gaining a deeper understanding of the molecular mechanisms governing GASP1-mediated GLP-1R trafficking could help in the development of improved therapies utilizing GLP-1R agonists to effectively combat T2D and obesity.
Abstract
Melanoma is an aggressive and lethal form of cancer with increasing incidence worldwide. In recent years, the FDA approved several promising treatments for malignant melanoma. However, ...treatment often fails due to the survival and regrowth of drug-resistant cells. Studies by our lab and others suggest that Melanoma Initiating Cells (MIC), a CD133+ population that forms xenograft tumors, may represent an intermediate stage in the acquisition of stable drug resistance. This study aims to determine the effect of CD133-expression and drug resistance in metastatic melanoma cells harboring difficult-to-treat mutation signatures by focusing on the two main downstream cascades of MAPK and PI3K/AKT/mTOR. Our results show that triple combination therapy using mebendazole (repurposed anthelmintic BRAF inhibitor), trametinib (MEK inhibitor) and metformin (repurposed anti-diabetic mTOR inhibitor) synergistically reduces cell viability of NRASQ61K melanoma cells when compared with mebendazole, trametinib or metformin alone. Further, the surviving fraction from each monotherapy is highly enriched for CD133+ cells. Thus, the multi-kinase inhibitor trametinib, along with repurposed anthelmintic and anti-diabetic drugs may inhibit MAPK and PI3K/AKT/mTOR pathways, which could potentially be an effective therapy for the resistant subpopulation of melanoma initiating cells.
Citation Format: Maryam AbdusSamad, Anirudh Gaur, Cynthia M. Simbulan-Rosenthal, Edward C. McCarron, Dean S. Rosenthal. Combination therapy with mebendazole, trametinib and metformin eliminates recalcitrant NRASQ61K melanoma cells. abstract. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2501.
Abstract
Melanoma is an aggressive and lethal form of cancer, responsible for 9,710 deaths in the US every year, as indicated by the Surveillance Epidemiology and End Results (SEER) program. Although ...early stages are surgically treatable, the advanced or metastatic stages are the most fatal, presumably due to resistance to chemotherapy, radiation or other therapies. Refractoriness of metastatic melanoma is due to self-renewal properties of a subpopulation of resistant cells, which may be the same as what have been termed “melanoma initiating cells” (MIC), many of which have been associated with the expression CD133. CD133 (PROM-1) is a 5-transmembrane-glycoprotein that is a cell surface marker for different cancers including late-stage cutaneous melanoma. By magnetic activated cell sorting, we isolated a CD133-positive population derived from a patient harboring a difficult-to-treat NRASQ61R/BRAFWT profile. The MIC were transfected with CD133-specific small-interfering RNA (siRNAs) to deplete CD133 protein levels in cells. Our results show that knocking down CD133 in NRASQ61R/BRAFWT mutant melanoma renders cells more sensitive to clinically employed-MEK/BRAF inhibitors. Thus, specific gene knockdown or small molecule targeting, along with the multikinase inhibitors, may be a high potency therapeutic of late-stage and recurrent melanoma. Analyses of patient-derived melanomas harboring different mutation signatures are currently in progress to determine if CD133 and MIC play broader roles in melanoma chemoresistance.
Citation Format: Maryam AbdusSamad, Anirudh Gaur, Hengbo Zhou, John L. Zapas, Cynthia M. Simbulan-Rosenthal, Edward C. McCarron, Dean S. Rosenthal. CD133 knockdown sensitizes melanoma to kinase inhibitors. abstract. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4224. doi:10.1158/1538-7445.AM2015-4224
Abstract
Purpose: High-risk melanoma is a lethal cancer with a high recurrence rates and variable disease-free intervals, due to tumorigenic cell populations that are resistant to treatment. Prominin ...1 (CD133) is a putative stem cell marker in a number of cancers, based on its expression in cells that possess greater tumorigenicity, motility and colony-forming ability in different types of cancer. Furthermore, Prominin 1 is highly expressed in late-stage metastatic melanoma showing its potential as a marker of a resistant cell population. Trametinib and dabrafenib are targeted kinase inhibitors recently approved by the FDA, and now widely used for treatment of melanoma, while the anthelmintic drug mebendazole, a multikinase inhibitor, also exhibits promising anticancer potential. The purpose of this study was to test the relative resistance of CD133+ and CD133- cells to these three agents, as well as to explore potential mechanisms underlying any differences.
Design: Three melanoma cell lines with different kinase mutation profiles were exposed to increasing concentrations of trametinib, dabrafenib, or mebendazole alone, or in combination, either before or after separation into CD133-positive and CD133-negative populations. Cells were then assayed for cell viability and CD133-positivity. Microarray analysis was performed on CD133-positive and CD133-negative cells to explore potential differences in gene expression profiles.
Result: In all parental cell lines, the percentages of the CD133-positive cells increased significantly (P<0.05) after high-dose drug treatment. The CD133-positive, drug resistant cells showed reduced proliferation until after the drug was removed. Correspondingly, the presorted CD133-positive population exhibited significantly higher (P<0.05) IC50s both for single and multidrug treatment. Microarray analysis revealed a significant (P<0.001, >1.5-fold) difference in expression of 265 genes in CD133-positive cells, including putative oncogenes such as POU5F1, NPM1, NES and MYC. Also, 10 of 18 ABC transporter genes were significantly (P<0.05) up-regulated in the CD133-positive population.
Conclusion: Both purified and mixed population-derived CD133 positive cells are more resistant to the three kinase inhibitors. Upregulation of ABC transporter genes and/or oncogenes may contribute to the drug resistance of CD133-positive cells.
Citation Format: Hengbo Zhou, Maryam Abdussamad, Amani Alomari, Anirudh Gaur, Cynthia S. Rosenthal, John L. Zapas, Dean S. Rosenthal. CD133 is associated with resistance of melanoma to multikinase inhibition. abstract. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3891. doi:10.1158/1538-7445.AM2014-3891
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