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.

Keywords: RAB7A; chemoresistance; cisplatin; endocytosis; lysosome.

Figure 1

(A) Cells were labeled live with Lysotracker Red DND-99 and Cell Mask (green), with LysoSensor DND-160 (yellow) and with LysoSensor DND-167 (blue) or were fixed and immunostained with anti-LAMP1 and anti-RAB7A antibodies (green). Nuclei were labeled with 4’,6-diamidino-2-phenylindole (DAPI) (blue). Scale bar: 10 μm. (B) IF intensity and (C) lysosomal area were quantified by ImageJ software. Data represent the mean ± standard error (SE) (three independent experiments, ≥50 cells). (D,E) Relative protein abundance of LAMP1, RAB7A, ATP6V1G1 was assessed by Western Blot (WB) and quantified by densitometry normalizing against α-tubulin. (F) qRT-PCR was performed on 2008 and C13 cells and the amount of RAB7A mRNA was quantified relative to GAPDH. Data represent the mean ± SE of at least three independent experiments (* p ≤ 0.05 and ** p ≤ 0.01 *** p ≤ 0.001).

Figure 2

(A–C) MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenol tetrazolium bromide) and (D–F) Sulforhodamine B (SRB) and Trypan Blue (TB) assays were performed on control (SCR) and on RAB7Ai 2008, HeLa and A431 cells after treatment with cisplatin (CDDP) at the indicated concentrations. (G–I) C13 (mock) or C13 expressing HA-RAB7A cells, HeLa and A431 Pt control (mock) and expressing HA-RAB7A were either untreated or treated with CDDP at the indicated concentrations and cytotoxicity was analyzed with MTT assay. (J) C13 (mock) or expressing the HA-RAB7AQ67L mutant protein were either untreated or treated with CDDP for 24 h and subjected to MTT assay. (K–M) MTT assay was performed on control (SCR) and on RAB7Ai 2008, HeLa and A431 cells after treatment with CDDP for 48 h at the indicated concentrations. Each point represents mean ± SE of at least three independent experiments run in eight replicates. Statistical significance was measured by comparing values with values of control untreated cells. (* p ≤ 0.05, ** p ≤ 0.01 and *** p ≤ 0.001).

Figure 3

(A) Accumulation of platinum in 2008 and C13 cells was measured by Inductively Coupled Plasma-Atomic Emission Spectroscopy (ICP-AES) after treatment with 20 μM CDDP for 24 h. Obtained values were normalized on cell number. (B) Total amount of extracellular proteins released from 2008 and C13 cells normalized on total cell proteins. (C,D) Relative amounts of CD63, CD9 and CD81 were examined by WB and quantified. (E) Expression of CD63, CD9 and CD81 on extracellular vesicles (EVs) from A431 and A431 Pt cells. (F) Platinum accumulation in C13 (mock) or C13 expressing HA-RAB7A was measured by ICP-AES after 24 h of treatment with 20 μM CDDP. (G) Expression of CD9 and CD81 was evaluated on EVs from mock and HA-RAB7A transfected C13 cells. Data represent the mean ± SE. of at least three independent experiments. ** p ≤ 0.01 and *** p ≤ 0.001

Figure 4

RAB7A expression was evaluated on protein extract from tissue tumor specimens of patients with ovarian cancer disease before (Pre) and after (Post) chemotherapeutic treatment. Levels of RAB7A protein was normalized on total Ponceau S protein staining.

Figure 5

Proposed chemoresistance mechanism mediated by RAB7A. CDDP enters the cell through specific transporters, by passive diffusion or by endocytosis. The uptake of CDDP in cells with functional endocytic trafficking leads to activation of apoptosis following sequestration of drug in lysosomes and consequent Lysosomal Membrane Permeabilization (LMP) induction. Uptake of CDDP into cells in which RAB7A is downregulated with a reduction in the size, number and acidification of lysosomes, leads to CDDP efflux through the EVs and then to a resistant phenotype. EE: Early Endosome; LE: Late Endosome; MVB: Multivesicular Body.