Stress-induced Rab11a-exosomes induce AREG-mediated cetuximab resistance in colorectal cancer

Exosomes are secreted vesicles made intracellularly in the endosomal system. We have previously shown that exosomes are not only made in late endosomes, but also in recycling endosomes marked by the monomeric G-protein Rab11a. These vesicles, termed Rab11a-exosomes, are preferentially secreted under nutrient stress from several cancer cell types, including HCT116 colorectal cancer (CRC) cells. HCT116 Rab11a-exosomes have particularly potent signalling activities, some mediated by the Epidermal Growth Factor Receptor (EGFR) ligand, Amphiregulin (AREG). Mutant activating forms of KRAS, a downstream target of EGFR, are often found in advanced CRC. When absent, monoclonal antibodies, such as cetuximab, which target the EGFR and block the effects of EGFR ligands, such as AREG, can be administered. Patients, however, inevitably develop resistance to cetuximab, either by acquiring KRAS mutations or via non-genetic microenvironmental changes. Here we show that nutrient stress in several CRC cell lines causes the release of AREG-carrying Rab11a-exosomes. We demonstrate that while soluble AREG has no effect, much lower levels of AREG bound to Rab11a-exosomes from cetuximab-resistant KRAS-mutant HCT116 cells, can suppress the effects of cetuximab on KRAS-wild type Caco-2 CRC cells. Using neutralising anti-AREG antibodies and an intracellular EGFR kinase inhibitor, we show that this effect is mediated via AREG activation of EGFR, and not transfer of activated KRAS. Therefore, presentation of AREG on Rab11a-exosomes affects its ability to compete with cetuximab. We propose that this Rab11a-exosome-mediated mechanism contributes to the establishment of resistance in cetuximab-sensitive cells and may explain why in cetuximab-resistant tumours only some cells carry mutant KRAS. ![Figure][1] Graphical Abstract This study highlights a clinically relevant mechanism in which stress-induced Rab11a-exosomes carrying the EGFR ligand, Amphiregulin (AREG) transfer drug resistance between genetically distinct colorectal cancer cells. Resistance to cetuximab, an anti-EGFR therapy, can be passed via Rab11a-exosomes from drug-resistant KRAS-mutant cells to previously drug-responsive KRAS-wild type cells. Unlike soluble AREG, Rab11a-exosome-associated AREG competes with cetuximab to activate EGFR signalling and promote EGFR-dependent outcomes, such as growth. This mechanism may support the co-operative evolution of clonal heterogeneity during tumour progression. ### Competing Interest Statement The authors have declared no competing interest. * AG1478 : EGFR kinase domain inhibitor AREG : Amphiregulin ANOVA : Analysis of Variance BSA : Bovine Serum Albumin Cav-1 : Caveolin-1 CMS : Consensus Molecular Subtype CRC : Colorectal Cancer CTX : Cetuximab dUC : Differential Centrifugation DMSO : Dimethyl Sulfoxide EGFR : Epidermal Growth Factor Receptor ERK : Extracellular Signal-Regulated Kinase 1 ESCRT : Endosomal Sorting Complex Required for Transport EV : Extracellular Vesicle FBS : Foetal Bovine Serum Gln : L-glutamine HER1 : Human EGFR Related 1 HRP : Horse Radish Peroxidase IgG : Immunoglobulin G ILVs : Intraluminal Vesicles ITH : Intratumoral Heterogeneity ITS : Insulin, Selenium and Transferrin mTORC1 : mechanistic Target of Rapamycin Complex 1 MAPK : Mitogen-Activated Protein Kinase mCRC : metastatic CRC MSI : Microsatellite Instability MSS : Microsatellite Stability NTA : Nanosight Tracker Analysis n/a : not applicable PBS : Phosphate Buffered Saline SD : Standard Deviation sEV : small EV SEC : Size-Exclusion Chromatography Syn-1 : Syntenin-1 TBS : Tris-Buffered Saline TBST : TBS with 1% Tween-20 TEM : Transmission Electron Microscopy UCA-1 : Uroepithelial Carcinoma-Associated-1 [1]: pending:yes