Rab21, an Endocytic Rab GTPase, is Involved in Drug Resistance in Prostate Cancer Cells

Multidrug resistance associated protein 1 (MRP-1), is a drug efflux pump encoded by ABCC1 gene (Leslie et al., 2005; Chang, 2007). MRP-1 was first identified in chemoresistant lung cancer cell line, as over-expressed transporter (Cole et al., 1992). Later on, variable expression of MRP-1 was also reported in various human tissues, for instance; high expression levels of MRP-1 were found in heart, kidney, lung, placenta, testis, skeletal muscles and adrenal glands. Whereas, the lower expression levels were reported in colon, brain, intestine, and liver (Leslie et al., 2005; Bakos and Homolya, 2007; Flens et al., 1996). MRP-1 is expressed in basolateral membrane of the polarised epithelial cells, where it plays an important role in protecting cells against toxic metabolites and xenobiotics (Sharom, 2008). Unlike other ABC transporters, MRP-1 or ABCC1 is composed of three transmembrane domains (TMDs) and the two nucleotide binding domains (NBDs) (Cole, 2014). The additional atypical TMD0 in MRP1 is believed to play an important role in sub-cellular localisation (Westlake et al., 2004, 2005) as truncated MRP1, lacking atypical TMD accumulates in endosomes (Westlake et al., 2005; Eva et al., 1998). TMD0 is needed for cell surface expression of MRP1 and its loss accelerates the internalisation of MRP1 (Lu et al., 2015). However, the exact mechanisms of MRP-1 trafficking and degradation still remain unclear.

The drug efflux ability of MRP-1 reduces the treatment efficacy against different diseases including epilepsy, depression, and cancer (Chen et al., 2013; Bao et al., 2011; Diestra et al., 2003). In fact, MRP-1 over-expression is reported to play a key role in emergence of multidrug resistance in various cancers during chemotherapy (Sharom, 2008). Moreover, the over-expression of MRP-1 along with MDR1 in various cancers has been found to be associated with relapse and considerable reduction in survival of cancer patients (Alisi et al., 2013; Haber et al., 2006; Vulsteke et al., 2013; Winter et al., 2013; Zhang et al., 2012). MRP-1 is also a major factor in developing chemoresistance in prostate cancer (John et al., 2008). Over-expression of MRP1 was found in drug resistant prostate cancer cell lines (Zalcberg, 2000; Takeda et al., 2007) Its ability to extrude antiandrogen compounds also suggests its role in antiandrogen therapeutic failure (Matthew et al., 2003).

Rab GTPases are the key regulators of membrane trafficking and are involved in endocytic, exocytic, and recycling pathways. Human genome encodes more than 70 Rab proteins that function as molecular switches, by cycling between the active and the inactive states (Samantha et al., 2008). Rabs are localised at different intracellular compartments to perform specific functions. Together with their effector proteins Rab GTPases regulate trafficking pathways and determine the destinations of cargo proteins. Dysregulation of Rab proteins have been implicated in cancer progression by disrupting the regulatory mechanisms of membrane trafficking (Tzeng et al., 2016). For instance, over-expression of Rab5 in colon cancer cells resulted in translocation of ABCB1 from plasma membrane to intracellular compartments, suggesting the role of Rab5 in endocytosis of ABCB1 (Kim et al., 1997). Studies have shown that Rab21, a homolog of Rab5, is also involved in endocytic pathways (Opdam et al., 2000). Rab21 is known for its role of protein cargo sorting from early to late endosomes and lysosomes (Egami and Araki, 2008, 2009). However, the role of Rab21 in regulating the membrane trafficking of MRP-1 and its consequence on multi drug resistance (MDR) remains unclear.

Over-expression of MRP-1 in epirubicin resistant prostate cancer cell (PC3/Res) led us to investigate its involvement in drug extrusion. Here, we hypothesised that altered expression of Rab21 might be involved in MRP-1 mediated epirubicin resistance in PC3 cells.

The monoclonal anti-MRP-1 antibody (clone, QCRL-1 Cat No sc-18835) and the anti-Rab21 antibody (clone B16K, Cat No sc-81917) were obtained from Santa Cruz Biotechnology. Alexa Fluor 568 conjugated secondary antibody (Cat No A11031), goat anti mouse HRP conjugated secondary antibody (Cat No G21040) and SlowFade® Gold Antifade mounting media with DAPI were from Invitrogen. Rab21 siRNA (Cat No sc-76322) was obtained from Santa Cruz Biotechnology. Doxorubicin hydrochloride and epirubicin hydrochloride were obtained from Sigma Aldrich. Dulbecco’s modified eagle media and all other cell culture reagents were obtained from Thermofisher scientific.

Both epirubicin sensitive (PC-3/Wt) and resistant (PC-3/Res) prostate cancer cell lines were maintained in DMEM, with 10% fetal bovine serum, penicillin 100 units/ml and streptomycin, 100 ug/ml. Cells were maintained at 37°C with 5% CO2 in a humidified incubator. The epirubicin resistant PC-3 cells were maintained under 0.5 micro molar epirubicin. Cells were transfected with siRNA against Rab21 using LipofectamineTM 3000 reagent (Invitrogen) according to manufacturerʼs instructions and analysed 72 h post transfection.

RNA was isolated using GeneJET RNA Purification Kit and DNA contamination was removed by treating total RNA samples with DNase I (both from Thermo Scientific). cDNA was synthesised using RevertAid™ First Strand cDNA Synthesis Kit (Thermo Scientific) following manufacturer’s instructions. The quantitative RT-PCR was performed using Thermo Scientific’s Maxima SYBR Green qPCR Master Mix (2X). Primers used were

MRP-1F: 5’-AGACAGAGATGCGAACCACC -3’,

MRP-1R: 5’-GGAAGCACCAGGAAACCACT -3’, Rab21F: 5’-GGGAAAAGAGTAAACCTTGCCA-3’, Rab21R: 5’-TAAACTAAAATCGCTCCATTTGAAT-3’ B2MF: 5’-TGCTGTCTCCATGTTTGATGTATCT-3’, and B2MR: 5’-TCTCTGCTCCCCACCTCTAA

GT-3’. The relative mRNA level was calculated by delta delta Ct method, normalising against B2M reference mRNA using CFX manager software (Bio-Rad).

Cells were lysed with SDS sample buffer by heating at 95°C for 10min. Cell lysates were resolved using 12% SDS-PAGE at 0.1 × 106 cell equivalents per lane, and then transferred to PVDF membranes (Immobilon; Millipore). Immunoblotting was performed using mouse anti MRP-1 and anti Rab21 monoclonal antibodies along with goat anti-mouse horseradish peroxidase conjugated secondary antibody. LuminataTM Forte Western HRP Substrate (Millipore) was used for detection. Photographic films were exposed for appropriate time and developed using automated film developer by Konica Minolta. Films were scanned using backlit film scanner and protein band intensities were quantified by normalising against Ponceau S staining as a loading control using ImageJ software.

PC-3 wild type and resistant cells were grown on coverslips. For immunofluorescent detection of MRP-1, cells were washed with dPBS and fixed using 4% formaldehyde solution (made freshly using paraformaldehyde) for 20 min. Following fixation, cells were permeabilised using 0.1% Triton X-100 and blocked with 5 % FBS for one hour. Cells were treated with 1: 50 dilution of anti-MRP-1 antibody for 1 h, washed thrice with dPBS and incubated with 1: 1000 dilution of Alexa Fluor 568 conjugated goat anti-mouse antibody for 1 hr. Cells were washed again, air dried and mounted using DAPI containing mountant. Labelled cells were observed using Olympus BXP-51 fluorescence microscope.

For drug efflux assays; 0.5×106 cells/well were seeded on cover slips and allowed to grow. Cells were washed with dPBS and incubated with 5uM epirubicin/ doxorubicin at 37°C for 2 h. After incubation, cells were allowed to efflux in drug free media for 1 h at 37°C. Cells were washed again and fixed with 4% formaldehyde for 20 min. DAPI Slowfade mounting media was used to counter satin nuclei. Non saturating microscopic images were obtained and mean fluorescence intensities of drugs were analysed and quantified using ImageJ Software.

GraphPad Prism software was used to analyse data. An unpaired t-test was used to calculate the p-value. Results were confirmed by performing at least two independent experiments with three technical replicates each. All results are presented as mean ± standard error of mean.

To study the effects of Rab21 on MRP-1 mediated MDR in prostate cancer PC-3 cells, the MRP-1 expression was compared in epirubicin (sensitive PC-3/Wt) resistant (PC-3/Res) cell lines. Our data revealed that MRP-1 mRNA was highly over expressed in PC-3/Res cells as compared to PC-3/Wt cells (Fig. 1A), Similarly, protein level of MRP-1 was also found elevated in PC-3/Res cells when compared with PC-3/Wt cells (Fig. 1B). Increased expression of MRP-1 in PC-3/Res cells suggest the possible role of MRP-1 in the development of resistance against epirubicin in prostate cancer. To investigate the sub cellular localisation of MRP-1 in PC-3/Res cells, immunofluorescence analysis was performed using specific anti-MRP-1 antibody. In drug sensitive PC-3/Wt cells, expression of MRP-1 was not observed. While, in epirubicin resistant PC-3/Res cell line, MRP-1 was predominantly found in plasma membrane in addition to its cytoplasmic localisation (Fig. 1C). Increased expression and cell surface localisation of MRP-1 in resistant PC-3/Res cells compared to drug sensitive PC-3/Wt cells indicated the role of MRP-1 in mediating MDR.

To substantiate the role of MRP-1 in mediating MDR in PC-3/Res cell line, we performed drug efflux assays. Doxorubicin and epirubicin were used to quantify drug retention in PC-3/Wt and PC-3/Res cells. These drugs are fluorescent in nature and their uptake or efflux by cells can be quantified by observing cells under a fluorescent microscope. Post efflux images were used to quantify residual intracellular doxorubicin. As shown in Figure 2A, the PC-3/Res cells showed decreased retention of doxorubicin than PC-3/Wt cells. Quantification of fluorescent intensity of doxorubicin showed a significant reduction in doxorubicin in PC-3/Res cells (Fig. 2B). Similar results were observed using epirubicin as a substrate of MRP-1. Decreased intracellular retention of epirubicin was observed in PC-3/ Res cells than PC-3/Wt cells (Fig. 3A and B). These findings indicate that the over expression of MRP-1 confers MDR in prostate cancer cells through drug efflux mechanism.

To examine whether expression of Rab21 is a contributing factor in MRP-1 mediated epirubicin resistance in PC-3 cells, we performed quantitative RT-PCR and Western blot analysis to measure mRNA and protein expression levels of Rab21 in PC-3/Wt and PC-3/Res cells. As shown in Figure 4A, the mRNA expression level of Rab21 was higher in PC-3/ Res cells as compared to PC-3/Wt cells. Similar results were obtained after Western blotting using anti-Rab21 antibody (Fig. 4B). The protein band intensity was also quantified and normalised against total protein loading control using Image J Software. Results showed a significantly increased expression of Rab21 in PC-3 epirubicin resistant cells (Fig. 4C).

Rab21 was up-regulated in PC-3/Res cells as compared to PC-3/Wt cells, suggesting that increased expression of Rab21 might contribute to MDR. We next explored whether epirubicin resistance could be attenuated by knocking down of Rab21 using Rab21 siRNA. Western blotting was performed post siRNA transfection and expression of Rab21 was found significantly reduced in PC-3/Res (Fig. 4D). We further examined whether down regulation of Rab21 had any effect on total MRP-1 protein expression level. Western blotting performed using anti-MRP-1 antibody showed that total MRP-1 protein remained unchanged after Rab21 knockdown as compared to control untransfected PC-3/Res cells (Fig. 4D). To see the effect of Rab21 knockdown on MRP-1 mediated drug resistance, epirubicin efflux assay was performed. Results showed that down regulation of Rab21 decreased the epirubicin efflux in PC-3/Res cells (Fig. 5A). The fluorescence intensity of epirubicin was also quantified using ImageJ Software and results showed a significant increase in the residual epirubicin in Rab21 knockdown PC-3/Res cells after efflux (Fig. 5B). The loss of drug efflux ability after Rab21 knockdown despite of unchanged total MRP-1 level, suggests that Rab21 down regulation may reduce MRP-1 surface localisation in PC-3/Res cells.

MRP-1 was first identified in drug resistant lung cancer cells and later in drug resistant acute lymphoblastic leukaemia, neuroblastoma, breast cancer, non-small cell lung cancer, and prostate cancer (Cole, 2014; Munoz et al., 2007). MRP-1 is an important drug efflux pump that transport various anticancer drugs including anthracyclins, vincristine, methotrexate etc. In addition to its role in drug extrusion, MRP-1 also exports a variety of physiological important molecules. Unlike P-gp, MRP-1 recognises different substrates due to bipartite nature and the plasticity of the single substrate binding site of MRP-1 (Johnson and Chen, 2017).

In the present study, we found that MRP-1 expression was significantly increased in epirubicin resistant PC-3 cells, suggesting the role of MRP-1 in mediating MDR in prostate cancer cells. This role of MRP-1 is in agreement with previous studies where expression levels of MRP-1 are known to increase with the progression of disease and invasiveness in prostate cancer (Gregory et al., 1998). Moreover, over-expression of MRP-1 is known to confer chemoresistance in doxorubicin exposed prostate cancer cell lines (Zalcberg et al., 2000). The sub cellular localisation of MRP-1 in PC-3/Res cells is also consistent with previous studies where MRP-1 showed both plasma membrane and internal pools. MRP-1 is known to localise in prostasomes of drug resistant prostate cancer cells which serve as reservoir of MRP-1 for translocation to the plasma membrane (Goma et al., 2014).

Rab proteins are well known regulators of membrane trafficking (Stenmark, 2009). We have observed the differential expression of Rab21 between drug resistant and drug sensitive PC-3 cells. Rab21 is ubiquitously expressed protein (Opdam, 2000) predominantly localised to endosomes and known to play a key role in early endocytic pathways. Rab21 has been reported to regulate endosomal trafficking of integrins from where they can be recycled back to plasma membrane through exocytic or recycling Rabs (Pellinen et al., 2006; Jones et al., 2006). Down regulation of Rab21 in drug resistant PC-3 cells using Rab21 specific siRNA, resulted in reduction of MDR phenotype in epirubicin resistant PC-3 cells. Our findings suggested that up-regulation of endogenous Rab21 level by epirubicin resistant cells evolved as a contributing factor to favour cell surface localisation of MRP-1 and hence drug resistance. Further studies are needed to better understand molecular link between Rab21 and MRP-1 and to find out if Rab21 has a role in other types of MDR cancer cells.

Our findings reveal the importance of Rab21 in MRP-1 mediated multidrug resistance. Interruption in membrane trafficking of MRP-1 using Rab21 siRNA resulted in increased intracellular drug retention, providing a potential strategy to overcome MDR.

This study was supported by grant from Higher Education Commission Pakistan (NRPU/2014/4506) to Dr. Moazzam Ali.

The authors have declared no conflict of interest.