Abstract
Background/Aim: Mammalian target of rapamycin (mTOR) inhibitors represent the standard of care for metastatic renal cell carcinoma (RCC). However, treatment outcomes are relatively poor, suggesting a potential problem with tolerating mTOR inhibitors. The aim of this study was to establish everolimus-resistant sublines and to compare their molecular characteristics with those of their counterparts. Materials and Methods: Human-derived RCC, Caki-2, and 786-O cells were continuously exposed to everolimus at 1 μM, and the established resistant sublines were designated as Caki/EV and 786/EV, respectively. Cellular characteristics were compared between both cells. Results: Caki/EV and 786/EV cells showed a decrease in sensitivity to everolimus as well as other mTOR inhibitors. Expression of mTOR and its effectors exhibited no alteration in resistant sublines and their counterparts. However, phosphorylation of S6K, an index of mTOR activity, decreased in resistant sublines. PCR array analysis of mTOR signaling pathway-related factors indicated that the expression of INSR, TP53, and IGFBP3 increased in Caki/EV cells, whereas that of TELO2, HRAS, and SGK1 was up-regulated in 786/EV cells. The levels of DDIT4, DEPTOR, HIF1A, and PLD1 mRNAs decreased in both cell lines. Conclusion: The novel everolimus-resistant Caki/EV and 786/EV cells exhibited cross-resistance to other mTOR inhibitors and decreased mTOR activity. Furthermore, down-regulation of DDIT4, DEPTOR, HIF1A, and PLD1 may contribute to everolimus resistance.
In 2020, the age-standardized incidence rate of kidney cancer in Japan was 7.6 per 100,000. Globally, there were 431,288 [95% uncertainty interval (UI), 418,145.0-444,844.0] incident cases of kidney cancer, and the age-standardized incidence rate was 4.6 per 100,000 (1, 2). Renal-cell carcinoma (RCC) has a lower incidence than other types of cancer worldwide, but its incidence is slightly higher in Japan, although the reasons for this discrepancy are not known. RCC is one of the cancers most resistant to conventional cytotoxic chemotherapy, and the standard of care was limited to cytokine therapy with interleukin-2 or interferon-α prior to 2004 (3, 4). However, a better understanding of the molecular mechanisms involved in the pathogenesis of RCC has led to the development of new treatment options, such as the inhibition of the mammalian target of rapamycin (mTOR), which targets downstream signaling cascades, tumor metabolism, and direct inhibition of vascular endothelial growth factor (VEGF)-mediated signaling (3-5).
The treatment of metastatic RCC has significantly improved with the advent of agents targeting the mTOR pathway, such as temsirolimus and everolimus, which were approved by the Food and Drug Administration in 2007 and 2009, respectively (5). The clinical efficacy of mTOR inhibitors has been demonstrated in patients with advanced RCC, showing poor prognosis or tolerance to VEGF receptor tyrosine kinase inhibitors (6-9). However, the treatment outcomes of mTOR inhibitors, such as progression-free survival and response rates across all patients were low compared to other drugs (10), suggesting a potential problem tolerating therapy with mTOR inhibitors.
The aim of this study was to establish everolimus-resistant sublines and to compare their molecular characteristics with those of their counterparts. In the present study, everolimus-resistant RCCs, designated as Caki/EV and 786/EV cells, were generated by continuous exposure to 1 μM everolimus using human-derived non-metastatic RCC, Caki-2, and 786-O cells, respectively. Using these resistant cells, sensitivity to some molecular-targeted drugs was examined, in addition to the elucidation of the molecular characteristics of Caki/EV and 786/EV cells.
Materials and Methods
Chemicals. Everolimus was obtained from Selleck Chemicals, LLC (Houston, TX, USA). Axitinib, sunitinib, and temsirolimus were purchased from Sigma-Aldrich (St. Louis, MO, USA). Erlotinib and sorafenib were purchased from LKT Laboratories Inc. (St. Paul, MN, USA). Rapamycin was obtained from LC Laboratories (Woburn, MA, USA). 2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-1), and 1-methoxy-5-methylphenazinium methylsulfate (1-methoxy PMS) were acquired from Dojindo Laboratories (Kumamoto, Japan).
Cells and cell culture. Caki-2 and 786-O cells were obtained from DS Pharma Biomedical (Osaka, Japan) and Summit Pharmaceuticals International (Tokyo, Japan), respectively, and used as human non-metastatic RCC models. Caki-2 and 786-O cells were maintained in RPMI1640 (Invitrogen™, Thermo Fisher Scientific K.K., Tokyo, Japan) supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen™) and 100 IU/ml penicillin-100 μg/ml streptomycin (Invitrogen™). Cells were cultured in an atmosphere of 95% air and 5% CO2 at 37°C, and sub-cultured with 0.05% trypsin-0.02% EDTA (Invitrogen™) every 3 or 4 days.
Establishment of everolimus-resistant sublines. The clinically achievable plasma concentration of everolimus at a clinical dose of 10 mg was approximately 60 ng/ml (equivalent to approximately 0.06 μM). However, the present study tried to establish everolimus-resistant RCC through exposure to 1 μM of everolimus, since exposure to a concentration <1 μM did not significantly alter cell growth. Continuous exposure to everolimus was performed using previously described methods (11-13). In brief, Caki-2 and 786-O cells were cultured in RPMI1640 medium containing 1 μM everolimus. Three months later, the everolimus-exposed cells were designated as Caki/EV or 786/EV. Caki/EV and 786/EV cells were maintained in a similar manner to Caki-2 and 786-O cells, respectively, except that the medium contained 1 μM everolimus.
Assay for cell growth activity. The growth activity of Caki-2, 786-O, and their everolimus-resistant cells was evaluated based on their growth curves. Cells (500 cells/well) were seeded onto 96-well plates on day 0 in 100 μl of culture medium without everolimus, and the cell number was counted from day 0 to day 8. Cell number was evaluated using the WST-1 (tetrazolium salts) colorimetric assay based on the MTT assay (11-13). According to the manufacturer’s instructions, the culture medium was exchanged with 110 μl of medium containing WST-1 reagent solution (10 μl of WST-1+100 μl of culture medium). After 2 h, the absorbance was determined at 450 nm with a reference wavelength of 630 nm using a Spectra Fluor microplate reader (Tecan Group Ltd., Männedorf, Switzerland). Preliminary experiments demonstrated a good proportional relationship between the absorbance and cell number (300-20,000 cells/well) in each cell. The doubling time of each cell line in log phase was calculated using Eq. 1:
Eq. 1
where t1 and t2 represent the time of cell counting, and N1 and N2 represent the cell numbers at t1 and t2, respectively (14).
Assay for growth inhibition by molecular targeting drugs. The effects of molecular-targeting drugs on the growth activity of each cell line were evaluated using the WST-1 assay (11-13). For short-term exposure, cells (1,000 cells/well) were seeded onto 96-well plates in 100 μl of RPMI1640 medium without any drugs. After 24 h of pre-culture, the medium was aspirated off and exchanged for one containing a test molecular-targeting drug at various concentrations. After incubation for 72 h at 37°C, cell number was evaluated using the WST-1 colorimetric assay, as mentioned above. In case of long-term exposure, the cell density on seeding was 500 cells/well and the drug exposure time was 168 h. The other experimental conditions were the same as those for short-term exposure.
The 50% growth inhibitory concentration (IC50) of the drugs was estimated according to the sigmoid inhibitory effect model, E=Emax×[1-Cγ/(Cγ+IC50γ)], using the non-linear least-squares fitting method (Solver, Microsoft® Excel) (10-13). E and Emax represent the surviving fraction (% of control) and its maximum, respectively; C and γ represent the concentration in the medium and sigmoidicity factor, respectively.
Immunoblotting. Cells were seeded onto 60-mm dish and cultured in the absence of everolimus for the desired time. The culture medium was aspirated and the cells were quickly frozen using liquid nitrogen. Proteins in the total cell lysate were extracted from cells using cell lysis buffer (Cell Signaling Technology Inc., Danvers, MA, USA) in addition to 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 5 μg/ml leupeptin. Proteins were separated by 12% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis and electrotransferred to a polyvinylidene difluoride membrane (Hybond-P membrane, Cytiva, Tokyo, Japan). Subsequently, the blot was blocked in wash buffer (10 mM Tris, pH 7.5, 150 mM NaCl, and 0.05% Tween-20) containing 5% skim milk. The membrane was soaked overnight in wash buffer containing specific primary antibodies, followed by incubation with horseradish peroxidase-conjugated secondary antibodies for 1 h (15). Antibody-bound proteins were visualized by treating the membrane with enhanced ECL™ Prime western blotting Detection Reagent (Cytiva) prepared immediately before detection. Finally, blot images were acquired using ChemiStage 16-CC (KURABO Industries Ltd., Osaka, Japan). The membranes were then stripped and re-probed with another antibody. Rabbit anti-mTOR, TSC2, raptor, or Akt antibodies and anti-phosphorylated (anti-phospho)-S6 kinase (Thr389), and anti-p70 S6 kinase antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-rabbit horseradish peroxidase (HRP)-conjugated immunoglobulin G (IgG) and rabbit anti-β-actin antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA) and Sigma-Aldrich, respectively.
Polymerase chain reaction array. Cells were pre-cultured for 48 h, and total RNA was extracted using a GenElute™ Mammalian Total RNA Miniprep kit (Sigma-Aldrich). The polymerase chain reaction (PCR) array was conducted using the RT2 Profiler PCR Array (Catalog No. PAHS-098, Qiagen, Hilden, Germany) and three independent sample sets were analyzed. Total RNA (500 ng) was used for reverse transcription with an RT2 First Strand kit (Qiagen) and a thermal cycler (i-Cycler, Bio-Rad Laboratories, Inc., Hercules, CA, USA) as per the manufacturer’s instructions. The reverse transcription reaction was conducted in 10 μl of reaction buffer at 42°C for 15 min, and terminated by heating at 95°C for 5 s, followed by cooling at 4°C. Real-time PCR was performed using a 7500 Fast Real-Time PCR system (Applied Biosystems, Waltham, MA, USA) and RT2 SYBR Green Master Mix (Qiagen). The PCR reaction was performed at 95°C for 10 min, followed by 40 cycles at 95°C for 15 s, and 60°C for 1 min. Dissociation was initiated at 95°C for 15 s, followed by 60°C for 1 min, and 95°C for 15 s.
Statistical analysis. Comparisons between the two groups were performed using an unpaired Student’s t-test. Statistical analysis was performed using JMP® Pro 15.2.0. (SAS Institute Japan Ltd., Tokyo, Japan). A two-tailed p-value <0.05 (two-tailed) was considered statistically significant.
Results
Growth curves of Caki-2, 786-O, and their everolimus-resistant cells. The growth curves of Caki-2 and Caki/EV cells exhibited a log phase of cell growth within 1 d after cell seeding, and the logarithmic phase continued for at least 3 days afterwards (Figure 1A). The estimated cell doubling times of the Caki-2 and Caki/EV cells were approximately 20.2 and 17.6 h, respectively. Furthermore, similar findings were observed in 786-O and 786/EV cells, with estimated cell doubling times of 12.8 h and 14.4 h, respectively (Figure 1B).
Growth curves for Caki-2, 786-O, and their everolimus-resistant cells. Each cell line was seeded at 500 cells/well on 96-well plates on day 0. Cell number was evaluated using the WST-1 assay from day 0 to day 8. Panel A shows the growth curves for Caki-2 (○) and Caki/EV (●), and Panel B indicates those for 786-O (○) and 786/EV (●). Each point represents the mean±S.D. (n=4), and the error bars were included in the symbols.
Effects of 72-h exposure to molecular target agents on cell growth. The IC50 values for mTOR inhibitors in Caki-2, 786-O, and everolimus-resistant cells in the short-term (72 h) exposure experiments are shown in Table I. The IC50 values for everolimus, temsirolimus, and rapamycin in Caki-2 cells were comparable to those in Caki/EV cells, and similar findings were observed in 786-O and 786/EV cells.
Fifty-percent growth inhibitory concentration of mTOR inhibitors exposed for 72 h in Caki-2, 786-O, and their everolimus-resistant cells.
Effects of 168-h exposure to molecular target agents on cell growth. In the long-term (168 h) exposure experiments, the IC50 values for mTOR and tyrosine kinase inhibitors in Caki-2, 786-O, and everolimus-resistant cells are shown in Table II. The sensitivity of Caki/EV cells to everolimus, temsirolimus, and rapamycin decreased significantly, indicating approximately 40- to 90-fold greater resistance than that of Caki-2 cells. The 786/EV cells also exhibited approximately 3- to 105-fold resistance to mTOR inhibitors. In contrast, the IC50 values for axitinib, sorafenib, and sunitinib were comparable between everolimus-resistant cells and their counterparts. However, the sensitivity of Caki/EV and 786/EV cells to erlotinib decreased significantly compared to their counterparts, showing 6.1- and 1.7-fold resistance compared to Caki-2 and 786-O cells, respectively.
Fifty-percent growth inhibitory concentration of mTOR inhibitors and tyrosine kinase inhibitors exposed for 168 h in Caki-2, 786-O, and their everolimus-resistant cells.
Time courses of expression and activity of mTOR and its effectors. In everolimus-free medium, the expression of mTOR in Caki-2 and Caki/EV cells remained unchanged for up to 72 h (Figure 2A). However, in Caki-2 cells, the phosphorylation of p70 S6K, which is downstream of mTOR, decreased in a time-dependent manner, whereas no blots were observed in Caki/EV cells. The expression of the mTOR-associated molecules TSC2, raptor, and Akt did not exhibit remarkable changes in Caki-2 or Caki/EV cells. Similar results were obtained for 786-O and 786/EV cells (Figure 2B). Moreover, the variation in the phosphorylation of p70 S6K observed by immunostaining was comparable to that observed by immunoblotting (data not shown).
Immunoblotting of mTOR and its related signaling molecules in Caki-2, 786-O, and their everolimus-resistant cells. The culture medium was aspirated off, and cells were quickly frozen by liquid nitrogen. Proteins in the total cell lysate were separated using 12% SDS-PAGE and electrotransferred to a polyvinylidene difluoride membrane. The blot was blocked in a solution of wash buffer containing 5% skim milk, and antibody-bound proteins were visualized by treating the membrane with the enhanced ECLTM Prime Western Blotting Detection Reagent prepared immediately before detection, and the membranes were stripped and reprobed with another antibody. Blot images were acquired using ChemiStage 16-CC, and β-actin was used as a reference.
mTOR signaling polymerase chain reaction array analysis. The mRNA levels associated with the mTOR signaling pathway were analyzed using a PCR array (Figure 3A and B). A volcano plot showed up- or down-regulation of mRNA in Caki/EV cells compared with Caki-2 cells (Figure 3A). Three mRNAs were up-regulated two-fold or higher in Caki/EV cells, whereas eight mRNAs were down-regulated ≥ two-fold (Figure 3A). In 786-O and 786/EV cells (Figure 3B), a volcano plot showed up- or down-regulation of mRNA, as well as Caki-2 vs. Caki/EV cells. Although the types of mRNAs did not always coincide, three mRNAs were up-regulated two-fold or higher in 786/EV cells, whereas 18 mRNAs were down-regulated two-fold or higher (Figure 3B). However, the levels of DDIT4, DEPTOR, HIF1A, and PLD1 mRNAs decreased in both Caki/EV and 786/EV cells compared to their counterparts.
Volcano plot of mTOR signaling PCR array in Caki-2, 786-O, and their everolimus-resistant cells. Panel A and B show Caki-2 vs. Caki/EV cells and 786-O vs. 786/EV cells, respectively. The black line indicates a fold-change in gene expression of 1. The dotted lines indicate two-fold change in gene expression threshold. The dashed-dotted lines indicate the threshold (0.05) for the p-value of the t-test.
Discussion
Everolimus has a distinct characteristic applied in both cancer chemotherapy and transplantation therapy. In cancer chemotherapy, everolimus is currently used in second line therapy after tyrosine kinase inhibitors, but acquired resistance to mTOR inhibitors is considered a universal clinical problem for patients with cancer. The development of resistant cell lines may therefore be useful for identifying biomarkers to predict the clinical response to mTOR inhibitors and develop strategies to mitigate treatment resistance. Herein, novel everolimus-resistant RCC cell lines were established by continuous exposure to everolimus, and their molecular profiles were examined and compared with their counterparts, Caki-2 or 786-O cells, which are used as non-metastatic human RCC cell models.
The established resistant Caki/EV and 786/EV sublines exhibited 40.2- and 104.6-fold resistance to everolimus after 168 h exposure (Table II) but not 72 h (Table I), respectively. Meanwhile, the growth rates of Caki/EV and 786/EV cells were comparable to their counterparts and did not show a decrease in growth activity (Figure 1). Interestingly, exposure to everolimus for 72 h did not induce treatment resistance. In comparison, previous studies have demonstrated resistance to everolimus after even 24-48 h exposure (16-18). These results suggest that Caki/EV and 786/EV cells acquired drug resistance based on growth suppression effects, but not cytotoxic effects. Considering previous reports that evaluated the cytotoxic action of mTOR inhibitors, the novel everolimus-resistant RCC sublines have been established as dissimilar to the previously resistant cells, showing the activation of signal transduction pathways via mTORC2 (16) and increased mitotic activity (17, 18).
Following drug exposure for 168 h, Caki/EV and 786/EV cells exhibited resistance to everolimus as well as other mTOR inhibitors, temsirolimus, and rapamycin (Table II), showing a cross-resistance phenotype. Moreover, both Caki/EV and 786/EV cells exhibited resistance to erlotinib, but not to other tyrosine kinase inhibitors, including axitinib, sorafenib, and sunitinib. Almost all these drugs are substrates for ABCB1 (19-21), which is an ABC transporter responsible for multidrug resistance; however, cross-resistance to these drugs was not observed (Table II). Therefore, the mechanism of resistance in Caki/EV and 786/EV cells is thought to be mediated by mTOR-related signal transduction, rather than by pharmacokinetic-based mechanisms.
To clarify the detailed mechanism of resistance in Caki/EV and 786/EV cells, the expression levels of mTOR and its effectors were examined. The expression of mTOR in Caki/EV and 786/EV cells remained unchanged up to 72 h after exposure, even in everolimus-free medium, and was also comparable to that in their counterparts (Figure 2), suggesting no changes in mTOR expression in relation to the resistance to everolimus. Other representative mTOR effectors, tuberous sclerosis complex (TSC) 2, raptor, and Akt, also showed no alterations in any of these cell lines (Figure 2). Alternatively, the phosphorylation of p70 S6K, an index of mTOR activity, decreased in Caki/EV and 786/EV cells even in everolimus-free medium, unlike in Caki-2 and 786-O cells. This was also supported by immunostaining for phosphorylation of p70 S6K (data not shown), suggesting a decrease in the activity of mTOR downstream as one of the mechanisms for acquiring resistance to everolimus.
Therefore, the expression of mTOR signaling pathway-related factors was examined using a PCR array (Figure 3). Among the 84 mRNAs examined, a volcano plot showed up- or down-regulation of mRNA in both Caki/EV and 786/EV cells compared to those of Caki-2 and 786-O cells (Figure 3A and B). The expression of insulin receptor (INSR), tumor protein p53 (TP53), and insulin-like growth factor binding protein 3 (IGFBP3) was higher in Caki/EV cells than that in Caki-2 cells (Figure 3A), whereas the levels of telomere maintenance 2 homolog (TELO2), Harvey rat sarcoma viral oncogene homolog (HRAS), and serum/glucocorticoid regulated kinase 1 (SGK1) were up-regulated in 786/EV cells compared with those in 786-O cells (Figure 3B). The types of mRNAs up-regulated were different between Caki/EV and 786/EV cells, and thus, these molecules would not be common factors for acquiring resistance to everolimus.
Alternatively, the levels of DNA-damage-inducible transcript 4 (DDIT4), DEP domain-containing mTOR-interacting protein (DEPTOR), hypoxia inducible factor 1, alpha subunit (HIF1A), and phospholipase D1 (PLD1) mRNAs decreased in both resistant cell lines compared to their counterparts (Figure 3). The relationship between these factors and the mechanisms of resistance was not clarified in this study, but these factors may play significant roles in acquiring resistance to everolimus. DDIT4 is participated in the regulation of cell growth, proliferation, and survival via inhibition of the activity of mTOR complex 1 (mTORC1) (22). In Caki/EV and 786/EV cells, the decrease in the expression of DDIT4 may lead to an absolver of mTOR signaling inhibition, resulting in the activation of mTOR signaling. Moreover, DEPTOR is known to be a mTOR binding protein that normally functions to inhibit mTORC1 and mTORC2 pathways, and thus DEPTOR is considered being a growth inhibitor (23). In addition, mTOR and DEPTOR work in a reciprocal manner, each negatively regulating the expression of the other (24). Doan et al. (24) reported that reintroduction of DEPTOR in the von Hippel-Lindau tumor suppressor protein (VHL)-deficient clear cell RCC cells significantly enhanced the efficacy of the mTOR kinase inhibitor, and loss of DEPTOR confers resistance to second-generation mTOR kinase inhibitors. Therefore, down-regulation of DEPTOR in Caki/EV and 786/EV cells is considered contributing partially to everolimus resistance. Interestingly, it was also reported that a decrease in DEPTOR expression could induce epithelial-to-mesenchymal transition (25, 26). Further, PLD1 is an enzyme that produces the lipid second messenger phosphatidic acid, and is also an upstream component in the mTOR pathway (27). Down-regulation of PLD1 in Caki/EV and 786/EV cells suggested the suppression of mTOR downstream signaling, causing resistance to everolimus. In addition, it was reported that cofilin 1 (CFL1) regulated expression of PLD1, and HIF-1α directly bound to CFL1 promoter to activate its transcription (28). These suggest the correlation with the down-regulation of PLD1 and HIF-1α, although a major route of HIF-1α translation was represented by the growth factor signaling pathway involving the downstream kinases PI3K/Akt/mTOR.
In conclusion, the novel everolimus-resistant sublines Caki/EV and 786/EV were generated, and a decrease in downstream mTOR activity was found to participate in the mechanism of resistance in Caki/EV and 786/EV cells. DDIT4, DEPTOR, HIF1A, and PLD1 down-regulation may also be involved. These everolimus-resistant sublines will be useful for identifying biomarkers to predict the response to mTOR inhibitors in molecular-targeted chemotherapy.
Acknowledgements
The Authors would like to thank Editage (www.editage.com) for the English language editing.
Footnotes
Authors’ Contributions
Yuko Nakayama: Investigation, data curation, visualization, writing, and original draft preparation. Daichi Enomoto: Writing and editing. Kazuhiro Yamamoto: Visualization, data curation, writing, reviewing, and editing. Kohji Takara: Data curation, writing–reviewing and editing, supervision.
Conflicts of Interest
The Authors declare that no conflicts of interest exist in relation to this study.
- Received July 11, 2023.
- Revision received August 3, 2023.
- Accepted August 4, 2023.
- Copyright © 2023 The Author(s). Published by the International Institute of Anticancer Research.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
