30 research outputs found
Harnessing autophagy to overcome mitogenâactivated protein kinase kinase inhibitorâinduced resistance in metastatic melanoma
Background
Patients with malignant melanoma often relapse after treatment with BRAF and/or mitogenâactivated protein kinase kinase (MEK) inhibitors (MEKi) owing to development of drug resistance.
Objectives
To establish the temporal pattern of CD271 regulation during development of resistance by melanoma to trametinib, and determine the association between development of resistance to trametinib and induction of prosurvival autophagy.
Methods
Immunohistochemistry for CD271 and p62 was performed on human naevi and primary malignant melanoma tumours. Western blotting was used to analyse expression of CD271, p62 and LC3 in melanoma subpopulations. Flow cytometry and immunofluorescence microscopy was used to evaluate trametinibâinduced cell death and CD271 expression. MTS viability assays and zebrafish xenografts were used to evaluate the effect of CD271 and autophagy modulation on trametinibâresistant melanoma cell survival and invasion, respectively.
Results
CD271 and autophagic signalling are increased in stage III primary melanomas vs. benign naevi. In vitro studies demonstrate MEKi of BRAFâmutant melanoma induced cytotoxic autophagy, followed by the emergence of CD271âexpressing subpopulations. Trametinibâinduced CD271 reduced autophagic flux, leading to activation of prosurvival autophagy and development of MEKi resistance. Treatment of CD271âexpressing melanoma subpopulations with RNA interference and smallâmolecule inhibitors to CD271 reduced the development of MEKi resistance, while clinically applicable autophagy modulatory agents â including Î9âtetrahydrocannabinol and Vps34 â reduced survival of MEKiâresistant melanoma cells. Combined MEK/autophagy inhibition also reduced the invasive and metastatic potential of MEKiâresistant cells in an in vivo zebrafish xenograft.
Conclusions
These results highlight a novel mechanism of MEKiâinduced drug resistance and suggest that targeting autophagy may be a translatable approach to resensitize drugâresistant melanoma cells to the cytotoxic effects of MEKi
Exendin-4 stimulates autophagy in pancreatic β-cells via the RAPGEF/EPAC-Ca PPP3/calcineurin-TFEB axis
Macroautophagy/autophagy is critical for the regulation of pancreatic β-cell mass and its deregulation has been implicated in the pathogenesis of type 2 diabetes (T2D). We have previously shown that treatment of pancreatic β-cells with the GLP1R (glucagon like peptide 1 receptor) agonist exendin-4 stimulates autophagic flux in a setting of chronic nutrient excess. The aim of this study was to identify the underlying pathways contributing to enhanced autophagic flux.Pancreatic β-cells (INS-1E),mouse and human islets were treated with glucolipotoxic stress (0.5 mM palmitate and 25 mM glucose) in the presence of exendin-4. Consistent with our previous work, exendin-4 stimulated autophagic flux. Using chemical inhibitors and siRNA knockdown, we identified RAPGEF4/EPAC2 (Rap guanine nucleotide exchange factor 4) and downstream calcium signaling to be essential for regulation of autophagic flux by exendin-4. This pathway was independent of AMPK and MTOR signaling. Further analysis identified PPP3/calcineurin and its downstream regulator TFEB (transcription factor EB) as key proteins mediating exendin-4 induced autophagy. Importantly, inhibition of this pathway prevented exendin-4-mediated cell survival and overexpression of TFEB mimicked the cell protective effects of exendin-4 in INS-1E and human islets. Moreover, treatment of db/db mice with exendin-4 for 21 days increased the expression of lysosomal markers within the pancreatic islets. Collectively our data identify the RAPGEF4/EPAC2-calcium-PPP3/calcineurin-TFEB axis as a key mediator of autophagic flux, lysosomal function and cell survival in pancreatic β-cells. Pharmacological modulation of this axis may offer a novel therapeutic target for the treatment of T2D.Abbreviations: AKT1/protein kinase B: AKT serine/threonine kinase 1; AMPK: 5' AMP-activated protein kinase; CAMKK: calcium/calmodulin-dependent protein kinase kinase; cAMP: cyclic adenosine monophosphate; CASP3: caspase 3; CREB: cAMP response element-binding protein; CTSD: cathepsin D; Ex4: exendin-4(1-39); GLP-1: glucagon like peptide 1; GLP1R: glucagon like peptide 1 receptor; GLT: glucolipotoxicity; INS: insulin; MTOR: mechanistic target of rapamycin kinase; NFAT: nuclear factor of activated T-cells; PPP3/calcineurin: protein phosphatase 3; PRKA/PKA: protein kinase cAMP activated; RAPGEF3/EPAC1: Rap guanine nucleotide exchange factor 3; RAPGEF4/EPAC2: Rap guanine nucleotide exchange factor 4; SQSTM1/p62: sequestosome 1; T2D: type 2 diabetes; TFEB: transcription factor EB
The role of retinoic acid in the differentiation of neuroblastoma
SIGLEAvailable from British Library Document Supply Centre-DSC:DXN019080 / BLDSC - British Library Document Supply CentreGBUnited Kingdo