CD9 inhibition reveals a functional connection of extracellular vesicle secretion with mitophagy in melanoma cells

Abstract

Tetraspanins are often used as Extracellular Vesicle (EV) detection markers because of their abundance on these secreted vesicles. However, data on their function on EV biogenesis are controversial and compensatory mechanisms often occur upon gene deletion. To overcome this handicap, we have compared the effects of tetraspanin CD9 gene deletion with those elicited by cytopermeable peptides with blocking properties against tetraspanin CD9. Both CD9 peptide or gene deletion reduced the number of early endosomes. CD9 peptide induced an increase in lysosome numbers, while CD9 deletion augmented the number of MVB and EV secretion, probably because of compensatory CD63 expression upregulation. In vivo, CD9 peptide delayed primary tumour cell growth and reduced metastasis size. These effects on cell proliferation were shown to be concomitant with an impairment in mitochondrial quality control. CD9 KO cells were able to compensate the mitochondrial malfunction by increasing total mitochondrial mass reducing mitophagy. Our data thus provide the first evidence for a functional connection of tetraspanin CD9 with mitophagy in melanoma cells. K E Y WO R D S CD9, cytopermeable peptides, EV biogenesis, mitophagy, tetraspanin  INTRODUCTIONExtracellular Vesicles (EVs) have emerged as a potent mechanism of intercellular communication that can contain a complex message composed of several signalling factors, enzymatic activities and genetic material (Yáñez-Mó et al., 2015). They are present in all biological fluids, offering a great opportunity for non-invasive biomarker discovery in several clinical scenarios; and have therapeutic potential for a wide variety of diseases, including neurodegenerative disorders and cancer (Fais et al., 2016).Tetraspanins are commonly used as markers of EVs populations, being present on both microvesicles (EVs budding from the plasma membrane) and exosomes (EVs derived from late endosomal multivesicular bodies (MVB) compartments) (Andreu & Yáñez-Mó, 2014). However, no fundamental alterations in endosome biogenesis and maturation have been described in This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Lipid Profiles of Human Serum Fractions Enhanced with CD9 Antibody-Immobilized Magnetic Beads

“…CD9, a member of the tetraspanin superfamily, has a variety of biological activities and is considered to be functionally crucial [ 19 ]. CD9 is known to accumulate in EVs [ 22 , 23 ]. In this study, we focused on CD9 and investigated the components enriched by magnetic beads with an immobilized CD9 antibody.…”

Tetraspanin Cd9b and Cxcl12a/Cxcr4b have a synergistic effect on the control of collective cell migration

“…Close relations to CD9 are the tetraspanins CD81 and CD63, which have been shown previously to substitute for CD9 in some circumstances. Overexpression of CD81 partially rescued the infertility phenotype seen in CD9 KO mice [ 39 ] and it was recently demonstrated that CD9 deletion in human melanoma cells was quickly compensated by CD63 expression upregulation [ 40 ]. It would be interesting to perform RNA-seq or SWATH-MS on the cd9 dKOs to identify upregulated RNA and proteins respectively [ 41 ].…”

Tetraspanin Cd9b and Cxcl12a/Cxcr4b have a synergistic effect on the control of collective cell migration

“…Close relations to CD9 are the tetraspanins CD81 and CD63, which have been shown previously to substitute for CD9 in some circumstances. Overexpression of CD81 partially rescued the infertility phenotype seen in CD9 KO mice (Kaji et al 2002) and it was recently demonstrated that CD9 deletion in human melanoma cells was quickly compensated by CD63 expression upregulation (Suárez et al, 2021). It would be interesting to perform RNA-seq or SWATH-MS on the cd9 dKOs to identify upregulated RNA and proteins respectively (Lin et al, 2019).…”