Modulation of RAB7A Protein Expression Determines Resistance to Cisplatin through Late Endocytic Pathway Impairment and Extracellular Vesicular Secretion

Abstract

Background: Cisplatin (CDDP) is widely used in treatment of cancer, yet patients often develop resistance with consequent therapeutical failure. In CDDP-resistant cells alterations of endocytosis and lysosomal functionality have been revealed, although their causes and contribution to therapy response are unclear. Methods: We investigated the role of RAB7A, a key regulator of late endocytic trafficking, in CDDP-resistance by comparing resistant and sensitive cells using western blotting, confocal microscopy and real time PCR. Modulation of RAB7A expression was performed by transfection and RNA interference, while CDDP sensitivity and intracellular accumulation were evaluated by viability assays and chemical approaches, respectively. Also extracellular vesicles were purified and analyzed. Finally, correlations between RAB7A and chemotherapy response was investigated in human patient samples. Results: We demonstrated that down-regulation of RAB7A characterizes the chemoresistant phenotype, and that RAB7A depletion increases CDDP-resistance while RAB7A overexpression decreases it. In addition, increased production of extracellular vesicles is modulated by RAB7A expression levels and correlates with reduction of CDDP intracellular accumulation. Conclusions: We demonstrated, for the first time, that RAB7A regulates CDDP resistance determining alterations in late endocytic trafficking and drug efflux through extracellular vesicles.

Older Adults with Physical Frailty and Sarcopenia Show Increased Levels of Circulating Small Extracellular Vesicles with a Specific Mitochondrial Signature

“…Notably, exosomes derived by B-cells are characterized by the tetraspanin markers CD9 and CD81, while CD63 is absent [36]. A previous report by our group showed that RAB7A, a small GTPase and a master regulator of the late endocytic pathway, was able to modulate secretion of CD9-and CD81-positive exosomes [37]. The decreased expression of tetraspanin CD63 found in the present study may therefore be indicative of an altered late endocytic pathway [38], possibly suggesting disarrangements in late endocytic trafficking in PF&S.…”

Mitochondrial Signatures in Circulating Extracellular Vesicles of Older Adults with Parkinson’s Disease: Results from the EXosomes in PArkiNson’s Disease (EXPAND) Study

“…The identification of sEV type and the characterization of protein cargo were accomplished by Western immunoblotting, as described elsewhere [24]. Briefly, equal amounts (1.25 µg) of sEV proteins from PD patients and controls were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently electroblotted onto polyvinylidenefluoride (PVDF) Immobilon-P membranes (Millipore, Burlington, MA, USA).…”

Autophagy modulating agents as chemosensitizers for cisplatin therapy in cancer

“…Cisplatin is characterized with a great potential to induce autophagy and cisplatin-resistant cells to present both high level of basal autophagy and alterations in vesicular [14][15][16][17][18]. The latter includes elevated extracellular vesicles secretion, alteration in lysosomal size, number and localization as well as disrupted localization and functioning of endosomal recycling compartment.…”

“…Additionally, another study proved that cisplatin treatment induces EV release which serves as mediators of communication with tumor environment to induce invasiveness and drug resistance [19]. Of note, other studies found reduced expression of RAB5A and upregulation of RAB8A in cisplatinresistant cells [16].…”

“…Aberrant function and expression of the lysosomal H + -pump in cisplatinresistant cells result in decreased lysosomal acidification [17]. This not only results in deficient lysosomal enzymes processing but additionally alerts lysosomal localization, trafficking, fusion and promotes lysosomal drug accumulation and exocytosis [15][16][17][18] The latter process may serve as an additional rout of drug efflux as cisplatin may be actively transported into lysosomes by ATP5A [5,6]. Deficiency in lysosomal enzymes activity was also linked to decreased EGF degradation [17].…”