IMP1 Enhances Extracellular Vesicle Secretion in a Transformation‐Dependent Manner

Abstract

Background RNA binding proteins regulate protein expression through coordinated binding to a series of related transcripts referred to as an RNA operon. Previous work demonstrates context and tissue specific roles for the RNA binding protein insulin-like growth factor 2 mRNA binding protein 1 (IMP1) in the context of cancer, especially colorectal cancer (CRC). Recent studies have linked IMP1 to extracellular vesicles (EV) and demonstrated that Imp1 is a potential regulator of autophagy during intestinal repair in response to damage. The endosome, EV, and autophagy pathways are interrelated; however, a direct role for IMP1 in coordinating these pathways in the GI tract has not been explored and is the objective of this study. Hypothesis We hypothesized that IMP1 is a mechanistic link between the endosome and autophagy pathways in CRC. Methods Ribosomal profiling and RNA sequencing compared transcript and translational efficiency profiles between IMP1 null and IMP1-overexpressing CRC cell lines. EVs were assessed by nanoparticle tracking analysis, western blot, and electron microscopy in CRC cells and non-transformed, mouse intestinal enteroids. Effects of IMP1 on endosome, EV, and autophagy pathways were assessed by electron microscopy, immunofluorescence, and western blot. Results: EV, endocytic, and exosome-related pathways were the most significant differentially regulated pathways by gene ontogeny analysis of RNA sequencing data in IMP1 null vs IMP1-overpressing CRC cells (p= 1.78x10-10, 2.43x10-10). Over 42% of the changed transcripts were associated with the vesicle pathway and differentially regulated by IMP1 expression. We found that IMP1 increased EV secretion in HT-29 (4483±403 vs 1934±414, p=0.006) and SW480 (3604±399 vs 2293±464, p=0.049) CRC cells vs null controls. IMP1 modulated translational efficiency or protein levels of early endosome proteins, including EEA1, but did not alter LC3II or p62 levels. When autophagic flux was inhibited with BafilomycinA1, the ability of IMP1 to enhance EV release in CRC cells was magnified. By contrast, Imp1 overexpression in non-transformed enteroids did not enhance EV secretion. Conclusions: Our novel findings suggest that the effects of IMP1 expression on EV biology differ between non-transformed and CRC cells. In CRC cells, IMP1 plays an important role in EV secretion and enhances early endosomal pathway protein translation and expression. Our findings have implications for the development of novel early detection and therapeutic approaches in CRC where IMP1 is overexpressed.