Oncogenic effects of RAB27B through exosome independent function in renal cell carcinoma including sunitinib-resistant

Abstract

Exosomes are 40-100 nm nano-sized extracellular vesicles. They are released from many cell types and move into the extracellular space, thereby transferring their components to recipient cells. Exosomes are receiving increasing attention as novel structures participating in intracellular communication. RAB27B is one of the leading proteins involved in exosome secretion, and oncogenic effects have been reported in several cancers. In recent years, molecularly targeted agents typified by sunitinib are widely used for the treatment of metastatic or recurrent renal cell carcinoma (RCC). However, intrinsic or acquired resistance to sunitinib has become a major issue. The present study aimed to elucidate the role of RAB27B in RCC including sunitinib-resistant and its role in exosomes. Bioinformatic analyses revealed that high expression of RAB27B correlates with progression of RCC. The expression of RAB27B protein in RCC cell lines was significantly enhanced compared with that in normal kidney cell lines. Furthermore, RAB27B protein expression was enhanced in all of the tested sunitinib-resistant RCC cell lines compared to parental cells. Although no specific effect of RAB27B on exosomes was identified in RCC cells, loss-of-function studies demonstrated that knockdown of RAB27B suppressed cell proliferation, migration and invasive activities. Moreover, anti-tumor effects of RAB27B downregulation were also observed in sunitinib-resistant RCC cells. RNA sequence and pathway analysis suggested that the oncogenic effects of RAB27B might be associated with MAPK and VEGF signaling pathways. These results showed that RAB27B is a prognostic marker and a novel therapeutic target in sunitinib-sensitive and -resistant RCCs. Further analyses should improve our understanding of sunitinib resistance in RCC.

Fig 1. Clinical significance of RAB27B expression in RCC.

(A) Kaplan-Meier analysis using the OncoLnc dataset revealed that the high RAB27B expression group (Z-score > 0) had significantly lower OS than did the low RAB27B expression group (Z-score < 0) in both ccRCC (right panel, P = 0.00179) and pRCC (right panel, P = 0.00407). (B) Among the ccRCC cohort of TCGA, the expression levels of RAB27B were significantly increased in pathological T4 category (right panel) and pathological G4 cases (left panel).

Fig 2. Analysis of RAB27B protein expression in RCC cells.

RAB27B protein expression levels in RCC cell lines, SU-R-RCC cell lines and HK2 cells were determined by Western blotting and densitometric analyses. The expression levels of RAB27B protein in RCC cell lines were elevated in comparison with that in HK2 cells. Furthermore, in all of the sunitinib-resistant RCC cell lines, the expression of RAB27B protein was enhanced compared to their parent cell lines. β-actin was used as a loading control.

Fig 3. Analyses of knockdown efficiency of si-RAB27B.

(A) RAB27B mRNA expressions were measured by RT-qPCR to validate the knockdown efficiency of si-RAB27B. The expression levels of RAB27B mRNA were effectively downregulated in the si-RAB27B-transfected RCC cells (**P < 0.001). (B) The expression of RAB27B protein were measured by western blot analyses. RAB27B protein levels were also downregulated in the cells transfected with si-RAB27B.

Fig 4. Oncogenic effects of RAB27B in RCC cells.

(A) Cell proliferation was examined using XTT assays in cells after RAB27B knockdown, revealing a significant inhibition compared with the control groups. (B) Wound healing assays revealed suppressed migratory activity in si-RAB27B transfected 786-o and A498 cells. (C) Matrigel invasion assays indicated that the number of invading cells was significantly decreased by RAB27B knockdown in 786-o and A498 cells. (**, P < 0.001).

Fig 5. Oncogenic effects of RAB27B in sunitinib-resistant RCC cells.

(A) Cell proliferation assessed by XTT assays was reduced in si-RAB27B-transfected sunitinib-resistant RCC cells. (B) Cell migration activity was suppressed by RAB27B knockdown in SU-R-786-o and SU-R-A498 cells. (C) Matrigel invasion activity was significantly decreased by si-RNA27B transfection of SU-R-786-o and SU-R-A498 cells. (**, P < 0.001; *, P < 0.01).