Background
Resistance to tyrosine kinase inhibitors (TKIs) is a concerning phenomenon for renal cell carcinoma (RCC) patients. RCC is among the top 10 cancers in the USA and 16th worldwide, being clear-cell RCC (ccRCC) the most prevalent histological subtype (> 80%) [
1,
2]. Patients with localised tumor usually undergo partial or radical nephrectomy, but approximately 30% of the patients present with de novo metastatic disease (mRCC) [
3]. RCC is a highly vascular tumor and sunitinib is the most commonly used anti-angiogenic targeted agent that acts by inhibiting receptor tyrosine kinases in endothelial cells. However, this study and other previously published research demonstrate the direct effect of sunitinib on cancer cells [
4‐
6]. Current treatment decision for mRCC is purely based on the clinical features: low risk patients are usually treated with anti-angiogenic tyrosine kinase inhibitors (TKIs) and intermediate/severe risk patients with immunotherapy [
7,
8]. However, initial responders to therapy will eventually develop resistance to TKIs within 10–14 months [
3,
9]. The consequence of treatment failure in patients is deleterious due to the development of senescent phenotypes that contribute to tumor progression on therapy withdrawal [
10]. Moreover, designing treatment strategies to overcome TKI resistance is challenging due to the lack of mechanistic insights and the availability of targeted therapies.
Y-box binding protein 1 (YB-1), a member of the cold-shock protein superfamily encoded by
YBX1 gene, is drastically increased in several types of cancer and it controls numerous cellular processes including DNA repair, transcription and translation of proteins [
11‐
13]. Recently, it has been shown to have an association with pathogenic stages in RCC and metastasis [
14,
15]. Furthermore, YB-1 has been involved in the “cross-talk” between mesangial and immune cells in inflammatory glomerular disease [
16]. This could be a critical finding given immunotherapy’s role in the intermediate/severe risk mRCC patients [
17‐
19].
On the other hand, ATP-binding cassette sub-family B member 1 (ABCB-1), plays a role in drug-resistance development in several cancers [
20,
21]. This transporter has been shown to modulate cancer stem cell-like properties and epithelial–mesenchymal transition in non-small cell lung cancer [
22]. In central nervous system, ABCB-1 upregulation restricts brain accumulation of dasatinib (TKI) limiting its effect in the patients [
23].
Therefore, this study investigated the function of YB-1/ABCB-1 in acquired sunitinib-resistance development in mccRCC. Herein, we confirm the direct effect of sunitinib in cancer cells as well as demonstrate the association between YB-1 and ABCB-1 in sunitinib-resistance development in metastatic clear-cell RCC (mccRCC). We also propose a combination therapy to re-sensitize resistant mccRCC to sunitinib. Overall, this study reveals a possible mechanism of sunitinib-resistance development and a potential treatment strategy to improve survival in resistant mccRCC patients.
Methods
Cell culture and patient tissue samples
De-identified mccRCC tissue samples were obtained from patients after receiving informed consent in Vancouver General Hospital (H09–01628). Primary kidney tumor specimens from mccRCC patients with or without sunitinib treatment were considered for further analysis. Each group had more than 5 patient samples. Caki-1 (ATCC, VA, USA) was grown in McCoy’s 5A media (Gibco, MD, USA) supplemented with 10%FBS (Hyclone, UT, USA). 786-O (ATCC, VA, USA) was grown in RPMI media (Gibco, MD, USA) supplemented with 10%FBS (Hyclone, UT, USA). Human Umbilical Vein Endothelial Cells (HUVEC) from pooled donors (Lonza, GA, USA) were maintained in EBM-Plus Bulletkit (Lonza, GA, USA). Cells were passaged 0.25% Trypsin-EDTA (Gibco, MD, USA). Where appropriate, cell numbers were counted with Automated Cell Counter TC20 (Bio-Rad, WA, USA). All cells were incubated at 37 °C in 5% CO2.
Reagents
The following reagents were purchased for this study: Sunitinib malate (Sutent, LC Laboratories, MA, USA); Elacridar (Toronto Research Chemicals, ON, CA); Mitomycin C and LY294002 (Sigma-Aldrich, MO, USA); AZD5363 and AZD8186 (Selleckchem, TX, USA); SL0101 (Calbiochem, CA, USA) and INK128 (Cayman Chemicals, MI, USA).
Sunitinib-conditioned Caki-1 cell-line
Caki-1 DC cell-line was prepared from the parental Caki-1 as previously published [
24]. Briefly, parental Caki-1 cells were grown to 50% confluence and then exposed to 0.1 μM sunitinib containing media. After 3–5 days, the media was replaced with fresh media for 24–48 h (Caki-1 DC, cycle1). Cells that showed proliferation were exposed to 25% higher concentration. The sunitinib on-off exposure cycle was maintained until approximately 20 cycles. In between each cycle, cells were allowed 5–8 passages. Caki-1 DC of cycle 15–18 were used for this study. Sunitinib-conditioned 786-ODC was also prepared from parental 786-O following the same procedure.
Cell viability assay
Cells were seeded in 96-well plates at 4000 cells/well and incubated for 24 h. Different concentrations of drugs were added and media with DMSO ≤0.1% was used as control. After 72 h, treatment media was removed and MTS reagent (Sigma-Aldrich, MO, USA) in fresh media was added (1:20 ratio). The cells were then incubated at 37 °C, in 5% CO2, and plate readings were taken at 30 min and 1 h at 490 nm (BioTek, VT, USA). Each experiment had 3 technical replicates and the experiments were repeated at least 3 times.
Scratch assay
Cells were allowed to grow to 80–90% confluence and, on the day of the experiment, were treated with 10 μg/ml of Mitomycin C for 2 h. The cells were scratched in a straight line with a sterile p200 tip, debris were removed by washes with phosphate-buffered saline (PBS; ThermoFisher Scientific, MA, USA), followed by incubation with appropriate cell medium. Images at time points and at matching reference coordinates were taken by an Axiovision microscope (Zeiss, ON, CA). Experiments were repeated at least 3 times.
Silencing of YB-1
Knockdown of YB-1 in Caki-1WT/DC was carried out using esiYB-1 and non-specific esiEGFP was used as control (Sigma-Aldrich, MO, USA). The cells were transfected with RNAiMax transfecting reagent (ThermoFisher Scientific, MA, USA) by reverse transfection method. Briefly, a master mix of RNAiMAX reagent was prepared in OptiMEM media (ThermoFisher Scientific, MA, USA) at 4:1 siRNA to reagent ratio. Aliquots of only esiYB-1 and esiEGFP (SCR) were also prepared in OptiMEM media. The two preparations were gently mixed and incubated at room temperature. Meanwhile, cells were enzymatically detached, counted and reconstituted in OptiMEM media. Complexes were then gently added to the reconstituted cells and plated to a final concentration of 5 μM for esiYB-1 and SCR. After 48 h post-transfection, fresh OptiMEM media was added and after 72 h post-transfection, the cells were harvested. Experiments were repeated at least 3 times.
Western blot
Western blots were carried out as previously published [
25,
26]. Primary antibodies were incubated overnight at 4 °C: YB-1 (ENZO Life Sciences, NY, USA) at 1:1000 dilution, P-Glycoprotein (ABCB1) rabbit monoclonal (Abcam, MA, USA) at 1:500 dilution, P-Akt (S473), β-Catenin, GSK-3β, SOX2 and GAPDH (Cell Signalling, MA, USA) at 1:1000 dilution. Secondary antibodies were horseradish peroxidase (HRP) conjugated anti-mouse and anti-rabbit antibodies (Cell Signalling, MA, USA) for use with SuperSignal West Femto Maximum Sensitivity Substrate (ThermoFisher Scientific, MA, USA) and imaged using autoradiography films (Genesee Scientific, CA, USA). Band intensity was quantified using ImageJ software (
NIH.gov). Experiments were repeated at least 3 times.
Quantitative RT-PCR
RNA was extracted from cell lines using RNeasy Mini Kit (Qiagen Hilden, DE), according to the manufacturer’s instructions. Taqman-primers used for qPCR included YB-1, ABCB-1 and GAPDH (ThermoFisher Scientific, MA, USA). Amplification was performed using a Viia7 qPCR (Applied Biosystems, CA, USA). Target gene expression was normalized to GAPDH levels and the comparative cycle threshold (Ct) method was used to calculate relative quantification of target mRNAs. Each experiment had 3 technical replicates and the experiments were repeated at least 3 times.
Immunofluorescence
Cells plated on coverslips (ThermoFisher Scientific, MA, USA) were allowed to grow for 48 h, fixed with 4% para-formaldehyde (Sigma-Aldrich, MO, USA), permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, MO, USA) and blocked with 2.5% horse serum (Vector Laboratories, CA, USA). Coverslips were incubated overnight at 4 °C with anti-P-Glycoprotein (ABCB-1) mouse monoclonal antibody at 1:100 (Sigma-Aldrich, MO, USA) and anti-YB-1 rabbit monoclonal antibody at 1:500 dilution. Secondary antibody staining was performed with anti-rabbit Alexa-594 and anti-mouse Alexa-488 (Invitrogen, CA, USA), mounted with DAPI (Vector Laboratories, CA, USA) and imaged with confocal microscope at 20X and 60X magnifications (Olympus FV3000RS). Experiments were repeated at least 3 times.
Immunohistochemistry
Formalin-fixed, paraffin-embedded tissue sections (4 μm) were deparaffinized by incubating slides at 60 °C for 1 h followed by repeated xylene and ethanol submersion. Antigen retrieval was performed with Diva Decloaker 10X (Cedarlane, ON, CA), steaming for 30 min, rinsed with dH2O and then incubated with 3% hydrogen peroxide (Sigma-aldrich, MO, USA). Sections were incubated with blocking from Vectastain ELITE ABC-Peroxidase kit according to manufacture’s protocol (Vector Laboratories, CA, USA). Slides were stained with rabbit monoclonal anti-ABCB-1 (1:100) and anti-YB-1 (1:500) antibodies overnight at 4 °C followed by secondary antibody staining using manufacturer’s protocol. Images were taken using SCN400 Slide Scanner (Leica Microsystems). Staining intensity was scored by a certified pathologist who was blinded to this study (score of 0–3). Cancer cells with positive staining in the tumor region were assigned an estimated percentage. Final intensity was calculated as: intensity = (score)×(percentage area)/100.
Proteomics
Tumors from sunitinib-sensitive and resistant rodents were used to obtain difference in protein expression pattern using Tandem Mass Tag (TMT) labeling of peptides as published before [
27]. For each group, tumor from 3 rodents were used.
Flow cytometry
Cells were non-enzymatically detached from the plated with Cell Stripper (VWR, Cat# CA4500–668) and stained with Annexin-V (APC, BD Bioscience, Cat#550475) at 1:100 dilution for 1 h. The cells were stained for DAPI (BD Bioscience, NJ, USA) at 1:1000 dilution for 15 min and analysed by FACSCanto II Flow Cytometry System (BD Bioscience, NJ, USA). Percentage of positively stained cell count was quantified using FlowJo_V10. Each experiment had technical duplicates and the experiments were repeated at least 3 times.
Engraftment of CAM tumor xenografts and imaging
Fertilized chicken eggs (Rudd, IA, USA) were incubated at 37 °C. On the fourth day of embryonic development (EDD-4), CAM assay was generated by transferring the egg contents into a plastic weight boat and incubated at 37 °C. On EDD-9, either Caki-1WT or Caki-1 DC cells were mixed with matrigel (BD Bioscience, NJ, USA) at 1 × 106 cells/10 μL and pipetted into the CAM. On EDD-11, tumor pictures and measurements were taken (pre-treatment) and the tumor bearing embryos were randomly divided into each treatment groups. Topical drug dosage was administered daily until EDD-18 (endpoint). The optical imaging for each CAM at pre-treatment and endpoint were taken using a Nikon SMZ18 stereo-microscope at 4X magnification and were digitized using an integrated Nikon DS-Ri2 digital camera (Nikon, TYO, JP). Tumor volume was measured using optical coherence tomography. Each group had more than 3 CAM tumor bearing embryos and the experiment was repeated 2 times.
Optical coherence tomography (OCT) imaging and analysis
OCT imaging was performed using a rapid 3D swept source Telesto 320C1 OCT system equipped with a telecentric scan lens (OCT-LK2) (Thorlabs Inc., NJ, USA). It has a center wavelength of 1300ηm, 3.0 μm axial resolution, 7.0 μm lateral resolution, a maximum imaging depth of 2 mm, and an A-line scan rate of 76 kHz. Imaging volumes of tumor bearing CAMs were acquired in a 5mmx5mmx2mm field of view at a resolution of 12μmx12μmx3.5 μm. Volumetric tumor image data.oct files were imported into MATLAB using code supplied by Thorlabs. Manual segmentation was performed on every tenth frame of OCT data using the MATLAB ‘imfreehand’ function to estimate for tumor volume. Changes in tumor volume were calculated by comparing the segmentation volumes from the pre-treatment and endpoint imaging datasets.
Tumor xenografts
Animal studies were performed as published before [
24] and in accordance to the guidelines of the Canadian Council on Animal Care with institutional certifications (University of British Columbia, A15–0231). Briefly, Caki-1WT/DC cells were injected subcutaneously (5 × 10
6 cells) in the flank region of 8 weeks old nude mice (Charles Rivers Laboratories, MA, USA). Mice were randomly divided into groups after the tumors reached a volume of 100-200 mm
3. Sunitinib malate was suspended in citrate-buffered solution (pH 3.5) and elacridar in diluent (0.5% methyl cellulose and 1% Tween-80 in ddH
2O). Treatment was administered by oral gavage once daily for 5 days followed by 2 days off for 2–3 weeks. For the combination treatment, mice were treated with elacridar 15 min prior to the administration of sunitinib malate. Tumor volume was measured every 3 days using calipers and calculated: tumor volume (mm
3) = length×width×height× 0.5. Each treatment group had more than 5 mice. Tumors were fixed with 10% para-formaldehyde (Sigma-Aldrich, MO, USA) for 24-48 h, 70% ethanol for 24 h (VWR International, PA, USA) followed by paraffin embedding.
Statistical analysis
The data are represented by mean ± standard error of mean (SEM). Mean was used as ‘centre value’ where appropriate. Samples were normalized to the experimental control to a value of 1.0 or 100%, where appropriate. Difference between two groups were calculated using analysis of variance with Student’s t-test, two-sided. Multiple comparisons were calculated with ANOVA, corrected with Tukey’s test. Cell viability assays were analysed for IC50 using non-linear regression for normalised response-viability slopes. The trend in tumor volume was measured with linear regression analysis. All the graphs were prepared and analysed using GraphPad Prism 8 software. A p < 0.05 was considered statistically significant and differences were denoted by asterisks (*p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001). All experiments were performed in triplicates and in three independent experiments.
Code availability
The association of the gene expression with patients’ survival outcome was calculated using the median overall survival time from TCGA cBioPortal. Median as “NA” was used when the median value was not obtained at 50-percentile. The log-rank test in the “survival” R-package was used to generate the Kaplan-Meier plots. Death of a patient was used as the censored event in the survival analysis.
Discussion
Among all the four histological sub-types, ccRCC is the most vascular tumor and the effective use of anti-angiogenic drugs like sunitinib are essential in improving patient outcomes [
36]. As sunitinib-resistance eventually develops in all treated TKI, it is imperative to gain insight into the molecular mechanism of acquired resistance to identify new treatments or methods to re-sensitize RCC. The effect of sunitinib on RCC is controversial with some data showing limited direct effect on RCC cells with anticancer effects due to inhibition of angiogenesis while other data suggests that sunitinib directly affects RCC cells [
5,
6]. Possibly, this apparent contradiction is due to the different cell-lines used in these studies. These cell-lines were derived either from primary tumor or of dubious histology [
37]. The current study, therefore, utilized Caki-1 cell-line, which is of metastatic origin with clear-cell RCC histology with wild-type VHL. We have also used another ccRCC cell-line, 786-O, that has VHL mutation.
We have established a sunitinib-conditioned in vitro
, ex vivo and sunitinib-resistant in vivo model, which resembles resistant mccRCC characteristics in patients [
24]. Our data show that endothelial cells (HUVEC) are sensitive to lower doses of sunitinib compared to Caki-1WT, but Caki-1 DC cells are tolerant to very high doses of sunitinib. Contrary to previous understanding, we show that sunitinib treatment significantly increases apoptosis and decreases proliferation in mccRCC cells providing evidence to a direct effect of sunitinib on cancer cells. To gain mechanistic insight into the differences between sunitinib-sensitive and resistant phenotypes, proteomics analysis on our in vivo model was performed. Our results indicate the involvement of ATP-binding cassette transporters, which are pivotal in drug resistance development in many cancers [
16,
38]. As YB-1 is an upstream regulator of many of these transporters, we have leveraged patient data from TCGA (cBioPortal). The analysis showed a marked decrease in median overall survival time in ccRCC patients with high YB-1 gene intensity compare to medium and low gene intensities. Moreover, in other cancer types, patients with mutations of YB-1 and ABCB-1 genes have decreased overall survival time. Recently, a study established the importance of YB-1 in mccRCC cell migration and adhesion by activation of nuclear factor kappa B (NF-κB) signalling pathway [
39]. Therefore, it is a logical extension to investigate the association of YB-1/ABCB-1 and sunitinib-resistance development in mccRCC tumors.
In our in vitro and in vivo models, we observed a significant increase in YB-1 and ABCB-1 protein and mRNA levels in the sunitinib-resistant samples compared to the sensitive samples. Moreover, knocking down YB-1 substantially downregulates ABCB-1 protein levels. This mechanistic insight is important because YB-1/ABCB-1 pathway is involved in survival, immune response, relapse and distant metastasis in patients. As a result, we tested numerous inhibitors against the common oncogenic pathways that are known to regulate YB-1 and ABCB-1 expression. However, drastic difference in ABCB-1 protein expression was only observed with very low dose of the dual mTOR inhibitor (0.5 μM INK128). Furthermore, a previously published study showed that YB-1 expression is regulated by mTOR pathway [
33]. This is intriguing because, once resistance develops, mTOR inhibitors are considered an option as the second-line of treatment for mccRCC patients [
34]. We show that the dual mTOR inhibitor (INK128) significantly reduced both YB-1 and ABCB-1 protein levels.
When treated with INK128, the change in mRNA level of YB-1 in Caki-1WT is not significant but is highly significant in Caki-1 DC. This could be explained by the effect of mTOR pathway in the translation of proteins and that inhibition of this pathway leads to accumulation of mRNA. Interestingly, the mRNA level of ABCB-1 remained non-significant in both Caki-1WT and Caki-1 DC. We have also observed no significant change in ABCB-1 mRNA level with siYB-1. These results suggest that mTOR pathway affects the translation of YB-1 protein but not of ABCB-1. It could be speculated that downregulation of YB-1 with either siYB-1 or mTOR inhibitor leads to increased ABCB-1 protein degradation, which is why a marked decrease in ABCB-1 protein level is observed but not in mRNA level. This mechanism of action of mTOR inhibitor could partially explain the success behind RECORD-3 clinical trial where a sequential treatment of sunitinib followed by everolimus had improved overall survival in patients [
40]. This sequential treatment strategy was simulated in our laboratory by re-challenging the sunitinib-conditioned Caki-1 DC cells with sunitinib after the dual mTOR inhibitor treatment and observed sensitization of Caki-1 DC using lower doses of sunitinib. For the first time, our study provides a possible mechanistic insight into the rationale use of mTOR inhibitors as a second-line of therapy in sunitinib-resistant mccRCC patients.
Unfortunately, it is well known that a combination of mTOR inhibitor and sunitinib is highly toxic in clinical trials, therefore, is not a clinically feasible option [
41]. On the other hand, an ABCB-1 blocker, elacridar, increases treatment efficacy in glioblastoma patients by overcoming blood-brain barrier [
42]. Moreover, a study on lysosomal sequestration of sunitinib in RCC, proposed the use of elacridar to increase the efficacy of sunitinib. However, the study did not elucidate the mechanism of increased ABCB-1 expression in sunitinib treated cells and used only in vitro model of 786-O cell line, which is of primary ccRCC origin [
43]. Therefore, the current study investigated the use of ABCB-1 inhibitor, elacridar, in mccRCC. Our data provides evidence that co-administration of sunitinib and elacridar significantly reduced cell viability in Caki-1 DC compared to sunitinib alone. To further support our hypothesis, we have generated chicken embryo chorioallantoic membrane (CAM) ex vivo tumor model with Caki-1WT and Caki-1 DC cells. In this assay, tumors engrafted with Caki-1WT (sunitinib-sensitive) significantly decreased in size when treated with either sunitinib alone or sunitinib/elacridar combination therapy. On the other hand, Caki-1 DC tumors (sunitinib-resistant), responded only to sunitinib/elacridar combination therapy. This observation was also confirmed in our in vivo model. Oral administration of 40 mg/kg of sunitinib significantly reduced the tumor size in Caki-1WT inoculated mice but had no effect on Caki-1 DC tumors. The Caki-1 DC tumors continued to grow despite increasing the dose of sunitinib (40 mg/kg to 80 mg/kg). However, the Caki-1 DC tumor size significantly reduced in the 40 mg/kg elacridar and 80 mg/kg sunitinib group treated compared to the 80 mg/kg sunitinib monotherapy treated group. This reduction in Caki-1 DC tumor size is also significant when compared to its matched pre-treated tumors. One limitation of our study is the use of immunocompromised models. As a result, the effect of YB-1/ABCB-1 pathway in pro-migratory immune cell mediated inflammation was not investigated. Hypothetically, the use of these inhibitors may also help in modulating tumor microenvironment in mccRCC tumors that could dictate response to immunotherapies.
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