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

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.


Introduction
Renal cell carcinoma (RCC) constitutes approximately 90-95% of all kidney neoplasms [1], making it the seventh most common site for tumours in 2013 [2]. The incidence of RCC has in RCC cells and sunitinib-resistant RCC cells were evaluated. The alteration of exosome secretion by knockdown of RAB27B and cell proliferative effects of exosomes derived from RAB27B down-regulated RCC cells were examined. Loss of function assays were performed in sunitinib-sensitive and -resistant RCC cells by examining cell proliferation, migration and invasion. The mechanisms of the effects of RAB27B were investigated by RNA sequencing and pathway analysis.

Analysis of the correlation between RAB27B and RCC
Kaplan-Meier and log-rank methods were used to analyze overall survival (OS) time using data in the OncoLnc dataset (http://www.oncolnc.org/), which contains survival data for 8,647 patients from 21 cancer studies included in TCGA. Also, OncoLnc is a useful tool for exploring survival correlations, and for downloading clinical data coupled to expression data for mRNAs, miRNAs, or long noncoding RNAs as previously described [36]. In order to evaluate the clinical relevance, a TCGA cohort database of 534 patients with ccRCC was used. Full sequencing information and clinical information were acquired using UCSC Xena (http:// xena.ucsc.edu/), cBioPortal (http://www.cbioportal.org/publicportal/), and TCGA (https:// tcga-data.nci.nih.gov/tcga/). The present study met the criteria for the publication guidelines provided by TCGA (http://cancergenome.nih.gov/publications/publicationguidelines).

Human RCC cell lines and cell culture
Human . SU-R-A498, SU-R-ACHN and SU-R-Caki1 were established by the same method. These cell lines were validated sunitinib resistance in xenograft assays. Parental and SU-R cells were subcutaneously injected into flanks of female nude mice (BALB/c nu/nu, 6-to 8-weeks-old, n = 4 for each group). After tumor formation was confirmed, we started gavage feeding of sunitinib (25mg/kg, five times a week). The tumors were harvested 20 days after injection. Comparison of the tumor volume of parental and SU-R cells were shown in S1 Fig. Human RCC cell lines were grown in RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Equitech-Bio, Inc., Kerrville, TX, USA), 50 μg/mL streptomycin, and 50 U/mL penicillin. For the experiments involving exosomes, exosome-depleted FBS (System Bioscience, LLC, Palo Alto, CA, USA) was used as a supplement in place of standard FBS. The HK2 cell line was grown in Keratinocyte Serum-Free Medium (Invitrogen) supplemented with 0.05 mg/mL bovine pituitary extract (BPE) and 5 ng/mL epidermal growth factor (EGF). These cell lines were maintained in a humidified atmosphere of 95% air/5% CO 2 at 37˚C.

RNA extraction and reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
Total RNA was isolated using Isogen (NIPPON GENE CO., LTD., Tokyo, Japan) according to the manufacturer's protocol, using SYBR-Green qPCR for RT-qPCR. First, 500 ng of total RNA was reverse transcribed into cDNA using the High Capacity cDNA Reverse Transcription kit (Thermo Fisher Scientific, Inc.) under the following incubation condition: 25˚C for 10 min, 37˚C for 120 min and 85˚C for 5 min. cDNA was used for q-PCR performed with the Power SYBR Green Master Mix (cat. no. 4367659; Applied Biosystems, Foster City, CA, USA) on a 7300 Real-Time PCR System (Applied Biosystems). The specificity of amplification was monitored using the dissociation curve of the amplified product. All data values were normalized with respect to glucuronidase β (GUSB), and the ΔΔCq method was used to calculate the fold-change. The following primers were used: RAB27B, forward primer, 5 0 -TAGACTTTCGGGAAAAACGTGTG-3 0 and reverse primer, 5 0 -AGAAGCTCTGTTGACTGGTGA-3 0 ; and GUSB, forward primer, 5 0 -CGTCCCACCTAG AATCTGCT-3 0 and reverse primer, 5 0 -TTGCTCACAAAGGTCACAGG -3 0 .

Transfection with small interfering RNA (siRNA)
As described previously [40], human RCC cells were transfected using Lipofectamine RNAi-MAX transfection reagent and Opti-MEM (both Thermo Fisher Scientific, Inc.) containing 10 nM siRNA. RAB27B siRNA (product ID, HSS184177 and HSS143561) or negative control siRNA (product ID, D-001810-10) (all Thermo Fisher Scientific, Inc.) to achieve loss-of-function. The knockdown efficiency of RAB27B siRNA was validated by confirming downregulation of RAB27B mRNA using RT-qPCR and RAB27B protein using western blotting.

Isolation and quantification of exosomes
Exosomes were purified by differential centrifugation procedures, as described previously [13,41]. Supernatants were collected from cells that had been cultured for 48 h in medium containing exosome-depleted FBS, and they were subsequently subjected to sequential centrifugation steps at 300g for 10 min, 2,000g for 10 min and 10,000g for 30 min to remove cell debris, dead cells and EVs other than exosomes. Supernatants were then centrifuged at 100,000g for 70 min at 4˚C (himac CP80WX, Hitachi, Ltd., Tokyo, Japan). The pelleted exosomes were suspended in PBS and collected by ultracentrifugation at 100,000g for 70 min. The purified exosomes were resuspended in PBS and used in subsequent experiments.
Exosome abundance was estimated with the ExoELISA-ULTRA CD63 kit (System Bioscience, LLC.) according to the manufacturer's protocol. This assay is a sensitive, direct Enzyme-Linked ImmunoSorbent Assay (ELISA) to quantitate exosome abundance in a given sample. The amount of exosomes is estimated by detecting CD63 on the exosome surface by specific antibody.

Cell proliferation, migration, and invasion assays
Human RCC cells were seeded in 96-well plates with 3x10 3 cells/well for XTT assays. After 72 h, cell proliferation was determined using a Cell Proliferation Kit II (Roche Diagnostics GmbH, Mannheim, Germany) as described previously [40].
Cell migration activity was evaluated with wound healing assays. Cells were plated in 6-well plates at 2x10 5 cells per well, and after 48 h of transfection the cell monolayer was scraped using a P-20 micropipette tip. The initial (0 h) and residual gap lengths 24 h after wounding were calculated from photomicrographs.
Cell invasion assays were performed using modified Boyden chambers consisting of Matrigel-coated Transwell membrane filter inserts with 8-μM pores in 24-well tissue culture plates (BD Biosciences, San Jose, CA, USA). 48 h following transfection, the cells were seeded in the upper chamber of 24-well plates at 1x10 5 /well with serum-free RPMI 1640 medium. RPMI containing 10% exosome-depleted FBS in the lower chamber served as the chemoattractant, as previously described [42]. 24 h after seeding, the cells that had passed through the pores and attached to the surface of the chamber were stained by Diff-Quick (a modified Giemsa stain) (Richard Allan Scientific, San Diego, CA, USA) and counted from photomicrographs.

RNA sequencing analysis and pathway analysis
Total RNAs from 786-o and A498 cells transfected with control siRNA or RAB27B siRNA were subjected to RNA sequencing, which was performed by RIKEN GENESIS CO., LTD., Tokyo, Japan. mRNA profiles were generated by NovaSeq 6000 (Illumina, Inc., San Diego, CA, USA).
Genes with significantly downregulated expression after transfection with RAB27B siRNA were compared with control siRNA (fold change < -1.0) and were then categorized with the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways through GeneCodis analysis (genecodis.cnb.csic.es) [43-45].

Statistical analysis
Data are presented as means ± standard deviation of at least 3 independent experiments. The relationships between two groups were analyzed using Mann-Whitney U tests. The relationships between three or more variables and numerical values were analyzed using Bonferroniadjusted Mann-Whitney U tests. All analyses were performed with Expert StatView software version 5.0 (SAS Institute, Inc., Cary, NC, USA). When P < 0.05, the data were accepted as showing a statistically significant difference.

Clinical significance of RAB27B expression in RCC
We first characterized the correlation between RAB27B expression and OS by performing a Kaplan-Meier analysis using the OncoLnc dataset. This analysis demonstrated that the group of patients with high expression of RAB27B (Z-score > 0) exhibited significantly lower OS rates compared with those in the low expression group (Z-score < 0) in both the ccRCC (P = 0.00179, Fig 1A, left panel) and pRCC (P = 0.00407, Fig 1A, right panel) cohorts. Next, we evaluated the correlations between RAB27B expression levels and patient clinicopathological parameters. Among the ccRCC cohort of TCGA, we found that the expression levels of RAB27B were significantly increased in pathological T4 category (Fig 1B, left panel) and pathological high grade G4 cases (Fig 1B, right panel). There was no significant difference of RAB27B expression between normal tissues and ccRCCs/pRCCs (S2 Fig).
Then, RAB27B protein expression levels in RCC cell lines, sunitinib-resistant RCC cell lines and HK2 cells were evaluated by Western blot analyses. The expression levels of RAB27B protein in RCC cell lines were elevated in comparison with the levels in HK2. Furthermore, it was revealed that the expression of RAB27B protein in all of the sunitinib-resistant RCC cell lines was enhanced compared to their parent cell lines (Fig 2).

Functional investigation of RAB27B for exosomes in RCC
To analyze the function of RAB27B for exosomes, we employed A498 cells because of their high expression level of RAB27B. First, we investigated whether knockdown of RAB27B reduced the secretion of exosomes. RT-qPCR analyses indicated effective downregulation of RAB27B mRNA in the si-RAB27B-tansfected RCC cells ( Fig 3A). Western blot analyses revealed that RAB27B protein levels were also downregulated in the cells transfected with si-RAB27B ( Fig 3B).

PLOS ONE
Oncogenic effects of RAB27B in sunitinib-sensitive and -resistant RCCs A498 cells transfected with si-control or si-RAB27B were cultured in RPMI 1640 medium supplemented with exosome-depleted FBS. Cell culture conditioned media were collected 48 h after transfection, and exosomes in the medium were isolated by ultracentrifugation and resuspended in PBS. We measured the amount of exosomes isolated from 40 mL of conditioned medium with the ExoELISA-ULTRA CD63 kit. Although the quantity of exosomes in the medium of si-RAB27B-transfected cells tended to be less than that of si-control-transfected cells, there was no statistically significant difference between them (S3A Fig).
We evaluated the effect of exosomes on cell proliferation with the XTT assay. We initially checked the effect of A498 exosomes on their own proliferation. A498 exosomes were added into the medium of A498 cells seeded in 96-well plates, and cell proliferation was determined after 72 h. XTT assays revealed that exosomes derived from si-RAB27B-transfected cells had no significant effect on cell proliferation compared to those from mock and si-control transfectants (S3B Fig, left panel). Subsequently, we examined the effect on the proliferation of cells other than the cells from which the exosomes were derived. We obtained the same results in the experiment when we added A498 exosomes to 786-o cells (S3B Fig, middle panel) and SU-R-786-o cells (S3B Fig, right panel). Thus, no specific effect of RAB27B on exosomal function was revealed in RCC cells in regard to both quantity and function.

Oncogenic effects of RAB27B in RCC cells
Loss-of-function studies using si-RAB27B were conducted to investigate the functional role of RAB27B in RCC cells. XTT assays with 786-o, A498, ACHN and Caki1 cells demonstrated that cell proliferation was inhibited in si-RAB27B-transfectants in comparison with that in the

PLOS ONE
Oncogenic effects of RAB27B in sunitinib-sensitive and -resistant RCCs mock-or si-control-transfectants ( Fig 4A). Wound healing assays revealed that cell migration activity was also inhibited in si-RAB27B-transfected 786-o and A498 cells (Fig 4B). Similarly, Matrigel invasion assays revealed that the number of invading cells was significantly decreased in si-RAB27B-transfected 786-o and A498 cells (Fig 4C). From the above, the oncogenic effects of RAB27B in RCC cells were demonstrated.

Oncogenic effects of RAB27B in sunitinib-resistant RCC cells
Subsequently, we investigated the functional role of RAB27B in sunitinib-resistant RCC cells. Loss-of-function assays by transfection with si-RAB27B were performed in sunitinib-resistant RCC cells as well as parental cells. Cell proliferation assessed by XTT assays was reduced in si-RAB27B-transfected SU-R-786-o, SU-R-A498, SU-R-ACHN and SU-R-Caki1 cells (Fig 5A). Cell migration activity was suppressed in si-RAB27B-transfected SU-R-786-o and SU-R-A498 cells (Fig 5B). Cell invasion activity was also suppressed by si-RNA27B transfection in SU-R-

PLOS ONE
Oncogenic effects of RAB27B in sunitinib-sensitive and -resistant RCCs 786-o and SU-R-A498 cells (Fig 5C). These results indicated that high expression of RAB27B was associated with oncogenic effects even in sunitinib-resistant RCC cells.

Analysis of the mechanism of oncogenic effects of RAB27B
In order to investigate the mechanism of oncogenic effects of RAB27B, RNA sequence and pathway analyses were performed (DRA009693: https://ddbj.nig.ac.jp/DRASearch/ submission?acc=DRA009693). A heatmap of RNA sequencing data was shown in S4 Fig. Based on the RNA sequencing data, we found 1069 genes downregulated in 786-o cells transfected with si-RAB27B, and 436 genes downregulated in A498 cells transfected with si-RAB27B compared with each cell type transfected with si-control. Among the 185 genes common to both, the most downregulated 20 genes are displayed in Table 1.
Next, we used GeneCodis analysis to categorize the si-RAB27B-downregulated genes into KEGG pathways. The results revealed that these genes were contained in 24 pathways, including the Vascular Endothelial Growth Factor (VEGF) signaling pathway, which is the main   PLOS ONE extracellular environment as exosomes, where they function in a multitude of intercellular signaling processes [12,14,47]. RAB27A and RAB27B are thought to function in MVEs' docking to the plasma membrane [15,29]. Furthermore, RAB27B has been suggested to be involved in the transfer of MVEs to the plasma membrane [29,48]. Contrary to expectations, suppression of exosome release was not observed in cells transfected with si-RAB27B in the present study. Since exosome secretion has been reported to increase in environments that are not suitable for cell survival, including chemotherapeutic treatment, hypoxia, heat stress, etc.
[49], it is possible that the tumor-suppressive effects of si-RAB27B acted to promote exosome secretion. Further studies are necessary to elucidate the mechanism underlying RAB27B and exosome secretion in RCC.
Exosomes have a range of different roles in cancer and they can be used as diagnostic markers and predict therapeutic responses. They may also constitute targets in therapeutic applications. Chen et al. reported that PD-ligand 1 (PD-L1) on exosome surfaces released from metastatic melanoma cells suppressed tumor immunity, and circulating exosomal PD-L1 can be a response predictor of anti-PD-1 therapy [50]. Kamerkar et al. demonstrated that exosomes derived from mesenchymal cells artificially incorporated siRNA or short hairpin (sh) RNA specific for oncogenic KRAS suppressed cancer in a plurality of mouse models of pancreatic cancer [51]. In renal cancer, differential protein profiling in urinary exosomes [52] and miRNAs contained in serum exosomes [53, 54] represent potential diagnostic markers. In addition, exosomes released from renal cancer stem cells contribute to triggering angiogenesis at premetastatic niches in the lung [55]. Further on, exosomes containing carbonic anhydrase 9 (CA9), a cellular response to hypoxia, were released from hypoxic RCC cells, and they are suggested to enhance angiogenesis in the microenvironment, thereby contributing to cancer

PLOS ONE
Oncogenic effects of RAB27B in sunitinib-sensitive and -resistant RCCs In the present study, the relevance of RAB27B to exosome secretion was not identified. Nevertheless, knockdown of RAB27B showed inhibitory effects of cell proliferation as well as cell migration and invasion. Therefore, we hypothesized that another functional role of RAB27B might be involved in epithelial-mesenchymal transition (EMT). Even though the western blot analyses of several representative EMT markers showed no common alterations in the RCC cells, the protein expression levels of Vimentin and N-cadherin were faintly decreased in 786-O or SU-R-A498 cells after si-RAB27B transfection (S6 Fig), suggesting that RAB27B might partially contributed to the EMT process in RCC as was previously reported in breast cancer [58].
As well as our demonstration, the oncogenic effects of RAB27B have been reported in several types of cancer. RAB27B has been shown to regulate invasive growth in vitro and in vivo in estrogen receptor (ER)-positive breast cancer cell lines [30] and to be involved in osteosarcoma cell migration and invasion [59]. Furthermore, it was suggested that disposal of tumorsuppressive miRNA via exosome release through the function of RAB27B is associated with acquisition of metastatic properties in bladder cancer [31]. According to our analysis, the expression level of RAB27B was associated with poor prognosis in RCCs. It has been demonstrated that high expression of RAB27B is correlated with poor prognosis in hepatocellular carcinoma [60], colorectal cancer [61] and ovarian cancer [62], consistent with our results. Although hypomethylated RAB27B is reported to be a progression-associated prognostic biomarker in glioma [32], the regulatory mechanisms of RAB27B expression remain to be defined. Thus, further research on the mechanisms is important to better understand these processes.
In terms of acquisition of drug resistance, a number of studies have demonstrated the contribution of exosomes [63]. For instance, there are several reports indicating a relationship between chemoresistance and exosomes in prostatic cancer [64][65][66]. In neuroblastoma [67] and ovarian cancer, [68] exosomal transfer of miR21 between cancer cells and stromal cells was indicated to contributes to development of chemoresistance. Recently, it was also demonstrated that ALK in the exosomes secreted by BRAF inhibitor-resistant melanoma cells transferred drug resistance through activation of the MAPK signaling pathway in recipient cells [69]. Furthermore, in renal cancer, Qu et al. showed that long noncoding RNA transmitted by exosomes promoted acquisition of sunitinib resistance [70]. With respect to RAB27B, metabolic reprogramming mediated by RAB27B was verified to induce doxorubicin resistance in breast cancer cells [71], and it was found that RAB27B is involved in chemoresistance to cisplatin in pancreatic cancer [72]. Moreover, exosome transfer from stroma to cancer cells regulated by stromal RAB27B is involved in therapeutic resistance in breast cancer [73]. In our study, RAB27B showed oncogenic effects in RCC cell lines with sunitinib resistance. Since the expression of RAB27B protein in all of the sunitinib-resistant RCC cell lines was enhanced compared to the parental cells, RAB27B may have some involvement in sunitinib resistance acquisition.
In order to investigate the mechanism underlying the oncogenic effects of RAB27B and its relevance to sunitinib resistance, pathway analysis was performed. GeneCodis analysis demonstrated that the VEGF signaling pathway and the MAPK signaling pathway were downregulated in RAB27B-knockdown cells. VEGF plays an important role in pathological angiogenesis associated with tumor growth [74], and also act as an autocrine growth factor [75]. VEGF have long been regarded as a promising therapeutic target in RCC [76], and is a major target molecule of sunitinib. Ishibashi et al. reported that tyrosine kinase inhibitors (TKIs) treatment to 786-o cells enhanced the expression of IL-6 and VEGF, and suggested that combination therapy of IL-6 inhibitor and TKIs may overcome TKI resistance [77]. MAPK pathway, which play essential roles in cell differentiation, proliferation and survival, also has been reported to be possible therapeutic target in RCC [78]. In addition, Gao et al. demonstrated that treatment targeting the ERK/MAPK pathway suppressed sunitinib resistance [79]. In ER-negative breast cancer, RAB27B-mediated modulation of β-catenin and VEGF was reported [80]. Further, in pancreatic cancer, EVs released by upregulated RAB27B activated p38 MAPK [81]. From the above, RAB27B may be associated with activated VEGF and MAPK signaling. Although knockdown of RAB27B showed no common alterations of the expression pattern of VEGF-or MAPK-related proteins in the western blot analyses, Erk1/2 or p38 MAPK protein was faintly decreased in 786-O, A498, or SU-R-786-O after si-RAB27B transfection (S5 Fig), suggesting that RAB27B might partially contributed to accelerating MAPK pathway in RCC. Interestingly, pathway analysis also showed an association between RAB27B and neurological diseases such as Huntington's disease, Parkinson's disease and Alzheimer's disease (AD). In fact, there are several reports showing the involvement of exosomes in these diseases [82][83][84]. Additionally, the upregulation of some synaptic GTPases, including RAB27B, was detected in tissues from patients with higher degrees of AD, and aberrant synaptic trafficking was suggested to modulate the progression of AD [85].
In conclusion, the present study investigated the association between RAB27B, an exosome secretory protein, and RCC. Although specific effects of RAB27B on exosomes were not identified, the oncogenic effects of RAB27B in RCC cell lines were demonstrated. Furthermore, the oncogenic effects of RAB27B were also demonstrated in sunitinib-resistant RCC cell lines. RAB27B may be a novel therapeutic target for sunitinib-sensitive and -resistant RCC. In addition, further studies may provide new insights into improving our understanding of sunitinib resistance in RCC.