Addition of DHA Synergistically Enhances the Efficacy of Regorafenib for Kidney Cancer Therapy

Renal cell carcinoma (RCC) arises from the renal tubular epithelium (1, 2), is the most common malignancy of the kidney, and is the sixth most common cancer in the United States. In contrast to many other cancers, the incidence of RCC is increasing likely due to smoking as well as the increased prevalence of the metabolic syndrome in the Western world (3–7). When localized to the kidney, surgical resection is usually curative; however, once the cancer metastasizes, the survival statistics, even with currently available novel therapies, are dismal. Among nonsurgical treatments of RCC, the immune modulators were historically associated with a very low success rate (8), likely related to immune suppression in the tumor microenvironment, possibly through local generation of tryptophan metabolites (9, 10). Enter the era of targeted therapeutics, which has resulted in the discovery of drugs possessing antiangiogenic activity via abrogation of vascular endothelial growth factor (VEGF) and other tyrosine kinase receptor signaling pathways involved in tumor growth and angiogenesis (11–14). However, while these approaches represented a major advance in the field, they are unfortunately associated with a high level of resistance after 1 to 2 years of treatment (15, 16), and furthermore, some are linked with a troublingly high rate of systemic hypertension (17). Therefore, novel approaches are urgently needed to improve the efficacy of these drugs. In the current study, we examined the use of the tyrosine kinase inhibitors in combination with compounds that we hypothesized would attenuate tumor resistance.

Regorafenib is a second-generation multikinase inhibitor that blocks the activity of kinases involved in the regulation of oncogenesis (Ras/Raf/MEK/ERK) and tumor angiogenesis (VEGF-R1, VEGF-R2, and VEGF-R3; ref.13). This drug is a marked improvement over the first-generation compounds (e.g., sorafenib) due to its higher specific activity leading to greater pharmacologic potency (13). The antitumor activity of regorafenib has been demonstrated in a variety of preclinical models and is associated with its kinase inhibitory effects, which results in suppression of cell proliferation, induction of apoptosis, and inhibition of tumor angiogenesis (13, 18, 19), the last being a key area of investigation for therapies of highly angiogenic RCC (20). We have recently shown that these multikinase inhibitors block soluble epoxide hydrolase (sEH; ref. 21), a key enzyme that metabolizes bioactive lipids of inflammation (22). Because inhibition of sEH stabilizes these lipids, thereby prolonging their beneficial effects on angiogenesis and inflammation, we asked whether it is possible to capitalize on this enzymatic activity to enhance the salutary effects of these specific kinase inhibitors in RCC.

sEH hydrolyzes epoxygenated fatty acids generated by the P450 metabolism of omega-3 and omega-6 polyunsaturated fatty acids (PUFA). Among these PUFAs, sEH metabolizes epoxyeicosatrienoic acids (EET), which are P450 products of arachidonic acid (ARA), and epoxydocosapentaenoic acid (EDPs), which are also P450 products but derive from docosahexaenoic acid (DHA), to their less bioactive diols (diols of EETs and EDPs, dihydroxyeicosatrienoic acids, DHETs) and dihydroxydocosapentaenoic acids, DiHDPEs, respectively; Fig. 1; ref. 23). While EETs possess anti-inflammatory (24) and antihypertensive (25) properties, they have been shown to be proangiogenic (26–28), a property that can clearly be detrimental in the treatment of highly angiogenic tumors, such as RCC. In addition, recent studies have suggested that EETs can promote the progression of cancer (29, 30), while other studies have contradicted these findings (31). In contrast, EDPs, which are also stabilized by sEH inhibitors (Fig. 1), have the opposite effect on angiogenesis (32); hence, we focus on the DHA metabolites of sEH in this study.

We hypothesized that the sEH inhibitory activity of regorafenib will result in marked increases in the anti-angiogenic and anti-hypertensive EDPs, which will be enhanced in the presence of exogenously administered DHA, usually the most abundant component of dietary fish oil supplements. We now show that the combination of DHA and regorafenib causes a decrease in HuVEC cell invasion as a measure of tumor angiogenesis as well as synergistically decreasing cell viability across three human RCC lines. Furthermore, by using a xenograft model of RCC in athymic nude mice, we demonstrate a decrease in tumor mass in vivo associated with the expected target effects and plasma oxylipin changes. Thus, once validated in human studies, novel therapy based on the addition of the dietary supplement DHA to regorafenib has the potential to result in an enhanced therapeutic efficacy of this kinase inhibitor for treatment of advanced RCC.

Human umbilical vein endothelial cells (HuVEC; Lonza) were grown in endothelial basal medium (EBM-2) supplemented with growth factors. The RCC cell lines 786-O(VHL^−/−), Caki-1(VHL^+/+), and Renca (VHL^+/+) were obtained from the American Type Culture Collection and the Renal proximal tubule epithelial cells (RPTEC or “normal human kidney, NHK”) were a primary (i.e., non-immortalized) line acquired from Lonza, which were cultured in renal epithelial cell growth medium (REGM; Lonza). All ATCC and Lonza cell lines undergo extensive authentication tests during the accessioning process as described on their Web site; in addition, all cells were frequently tested for mycoplasma in the author's laboratory. The 786–0 and Caki-1 and Renca cells were maintained in RPMI, and NHK cells were grown and cultured in DMEM, both supplemented with 10% FBS, 100 units/mL streptomycin, and 100 mg/mL penicillin. Cells were maintained at 5% CO2 and at 37°C. All cell lines were used with a passage number of two and confirmed to be free of mycoplasma, per monthly laboratory testing.

All animal studies were approved by the University of California Davis Animal Use and Care Committee and were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Thirty-six 4-week-old male athymic nude Nu/Nu mice (Harlan Laboratories) were acclimated to housing conditions for 1 week and were kept under a 12-h light–dark cycle with free access to water and food for the duration of the experiment.

Subsequently, mice were injected a suspension containing 786-O cells at 0.5 × 10⁶ mixed in 30% of non-growth factor reduced Matrigel (Corning Inc.) subcutaneously in the flank region as previously described (33). Tumor growth was monitored twice a week for each mouse using a digital caliper. Tumor volume (mm³) was calculated as length*(width²/2). When tumor volume reached approximately 100 mm³ (around 3–4 weeks of inoculation), treatments and diets began.

Mice were randomly divided into two experimental dietary groups: control diet (5% corn oil) or a 1% DHA-enriched diet (17.5 g DHA and 52.5 g corn oil/kg). DHA ethyl ester replaced corn oil to retain equal dietary fat between both isocaloric diets. The detailed composition of the diets is described in Supplementary Table S1. Half of the mice in each dietary group were given a daily administration of either 10 mg/kg regorafenib or vehicle (PEG400/125 mmol/L aqueous methanesulfonic acid; 80/20) via oral gavage. Treatments continued for 3 weeks. Body weights and tumor sizes were measured every 2 days. At the end of the experiment, plasma and tissues were harvested for immunohistochemistry and oxylipin analysis.

HuVECs were grown in 24-well plates containing Transwell inserts of 8-μm pore polyvinylpyrrolidone-free polycarbonate filters coated with Matrigel on the upper compartment at a density of 1×10⁵ cells in EBM-2 media containing 0.1% BSA (34). EBM-2 media consisting of 10% BSA were added in the bottom compartment of the well as a chemoattractant. Both upper and lower chambers contained one of the following treatments: 1 μmol/L ARA, 1 μmol/L DHA, 1 mol/L DHA plus 1 μmol/L regorafenib, 1 μmol/L regorafenib, 1 μmol/L linoleic acid (LA) or DMSO. Cells were incubated at 37°C for 20 hours to allow for migration. Afterward, Transwells containing cells were washed in PBS, fixed in 5% glutaraldehyde, and stained with 0.5% Toluidine Blue. Next, the upper wells were gently scraped to allow for imaging and quantification of cells that had migrated toward the lower compartment of the Transwell inserts.

Cell viability was assayed by plating cells in 96-well plates at a density of 3 × 10³ cells. After 24 hours, NHK, 786-0, Caki-1, and Renca cells were treated with 1 μmol/L of the fatty acids LA, ARA, eicosapentaenoic acid (EPA), and DHA each with the presence or absence of 1 μmol/L regorafenib and DMSO control. After 24 hours of treatment, cells were quantified via hemocytometer and treated with media containing MTT solution (1 mg/mL thiazolyl blue tetrazolium bromide) for 3 hours. Afterward, the MTT solution was removed, and the blue crystalline precipitate internalized by the cells were dissolved with DMSO. Finally, plates were placed in a plate reader to measure visible absorbance at 570 nm.

HuVECs were grown at a density of 2 × 10⁵ cells in six-well plates. After serum starvation for 6 hours in EBM-2 media containing 0.1% bovine serum albumin (BSA), cells were treated with 1 μmol/L omega-6 LA, 1 μmol/L LA + regorafenib, 1 μmol/L DHA, or 1 μmol/L DHA + 1 μmol/L regorafenib for 24 hours. Cells were then lysed, and total cell lysates were analyzed for proteins of interest using antibodies against phosphorylated VEGFR-2 and β-actin (Cell Signaling Technology).

Immunoblotting of tumor tissue was performed as previously described (35). Briefly, after the indicated treatments, the tissues were washed with PBS, lysed, and subjected to immunoblotting. For the xenograft tissue tumors, proteins were extracted with T-PER. The membranes were blocked in 5% nonfat dry milk for 1 hour at room temperature, incubated with antibodies (β-actin, pVEGFR-2, VEGFR-2, pERK1/2, and ERK1/2), and then probed with HRP-tagged anti-mouse or anti-rabbit IgG antibodies. The signal was detected using ECL solutions (Thermo Fisher Scientific). Densitometry was performed using ImageJ software.

The quantitative profiling of oxylipin was carried out as previously described (36). Briefly, plasma samples were extracted using solid-phase extraction cartridges. Samples were eluted through the cartridges, dried, and then reconstituted by adding 200 nmol/L 1-cyclohexyl-dodecanoic acid urea (CUDA) methanol solution. Oxylipins were then detected using high-performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). The optimized conditions of chromatographic separation have been reported previously (37) as have the instrument parameters, including MRM transitions (ref. 36; Applied Biosystems, 4000 QTRAP tandem mass spectrometer).

All data were analyzed for significance in SAS version 9.3 (SAS Institute Inc.). Cell numbers from invasion assay, tumor weights, oxylipin quantification, and tumor volumes were analyzed for significance by one-way ANOVA at P < 0.05. Where significant differences were found, a Tukey multiple comparison test was performed at a probability of α = 0.05. The data are presented as means ± SEM. Different letters appearing above bars in bar graphs designate that significant differences were found, while bars sharing the same letter indicate that significance was not achieved. Bars having two letters (such as ‘bc’) indicate that significance was not achieved compared with group ‘b’ or group ‘c’.

Synergy was assessed by calculating the combination index (CI) values using CalcuSyn software, which provides a quantitative definition for additive effect (CI = 1), synergism (CI < 1), and antagonism (CI > 1) in combination treatments.

Because the addition of DHA in the presence of sEH inhibition provided by regorafenib would be expected to increase local EDP levels (Fig. 1) and thereby attenuate angiogenesis (32), we first evaluated this property in an in vitro model of angiogenesis (38, 39). Because we previously reported an increase in HuVEC proliferation and infiltration when treated with EETs, specifically 11,12-EET and 14,15-EET, which are generated from ARA (32), we utilized the omega-6 PUFA LA, the predominant PUFA found in corn oil, as an additional control. HuVEC were grown on matrigel in Transwell plates, in which cells that infiltrated the matrigel were enumerated in order to assay for invasive potential (see Materials and Methods). After treatment with 1 μmol/L ARA, 1 μmol/L DHA, 1 μmol/L DHA + 1 μmol/L regorafenib, 1 μmol/L regorafenib, 1 μmol/L LA or DMSO for 20 hours, invading HuVEC were imaged (Fig. 2A) and quantitated (Fig. 2B). The cells treated concurrently with DHA and regorafenib were found to be the least invasive of all conditions tested, with a reduction of ∼60% compared with DMSO control. This combination likely resulted in a higher amount of EDPs, which comes about with a high availability of DHA in concert with the inhibition of sEH afforded by regorafenib. To confirm target inhibition by regorafenib, we evaluated its kinase activity on the phosphorylated (kinase-active) form of VEGFR-2 (40) and showed that pVEGFR-2 expression was lower in cells treated with regorafenib after both LA and DHA treatments alone, but more pronounced with the combination (Fig. 2C), thereby confirming target engagement with regorafenib. Regorafenib alone demonstrated a nonsignificant decrease in VEGFR-2 expression (data not shown).

These results are consistent with previous data showing that EDPs inhibit VEGF-induced cell migration in HuVEC after being treated with 19,20-EDP (32). Further, these findings demonstrate a synergistic effect of DHA and regorafenib on endothelial cells to suppress angiogenesis, primarily via suppression of endothelial cell migration likely via high levels of EDP.

We next assessed cell viability in vivo utilizing two human kidney cancer lines (786-0 and Caki-1) and the mouse kidney cancer cell line Renca, as well as primary (non-immortalized) normal human kidney epithelial (NHK) cells as controls. All cells were treated with 1 μmol/L of the fatty acids LA, ARA, EPA, and DHA each, in the presence or absence of 1 μmol/L regorafenib and DMSO control. LA, which is the major polyunsaturated fatty acid comprising corn oil, served as the in vitro control treatment that would best mimic the conditions of corn oil administration in vivo such that the two experiments could be compared. EPA was used to discern if the mitigation of cell viability was due to an omega-3 effect or specifically to DHA. After 24 hours of treatment, both regorafenib + DHA and DHA alone decreased cell viability in all three of the cancer lines with no significant effect on NHK cells; however, a greater decrease in cell viability was found with the former treatment (Fig. 3). Furthermore, cells were quantified from an experiment performed in parallel to the MTT assay on the four cell types (inset, Fig. 3); these data demonstrate that the combination of regorafenib with DHA resulted in synergistic responses after 24 hours of incubation. The therapeutic efficacy was assessed by calculating CI values using CalcuSyn software (41). Analysis of combination therapeutic indexes revealed synergistic effects by demonstrating the CI values in the range of 0.61 to 0.85 (synergy defined as CI <1) with the combination of regorafenib and DHA alone among the three RCC lines. Antagonistic interactions (CI>1) were found with LA and ARA with CI calculations 1.14 and 1.23, respectively. These findings demonstrate that the combination of regorafenib and DHA produced a synergistic decrease in several RCC, but not in normal renal epithelial cell, viability.

In light of previous data from one of our laboratories demonstrating that treatment with EDP concurrently with sEH inhibition attenuated both tumor growth and angiogenesis (32), we next asked whether the concurrent addition of regorafenib and DHA synergizes in an in vivo xenograft model of human RCC using the 786-0 (VHL^−/−) human RCC cell line used in several previous studies (40, 42, 43). Male athymic Nu/Nu mice were started on the diets and pharmacologic treatments after the 786-0 xenografts achieved a volume of ∼100 mm³. The mice were given free access to either a diet with fat originating from corn oil, which is naturally high in the omega-6 PUFA LA, or a 1% enriched DHA diet. The DHA concentration in the diet was determined by metabolic body size using an average daily food intake of 5 g/day/mouse, which translates to ∼3.1 g/day of DHA in a 70-kg human. This amount is achievable through consuming fish oil supplementation and in fact has been recommended to decrease progression in IgA nephropathy, a common renal disease (44).

Mice were given either regorafenib (10 mg/kg/day) or vehicle control administered by oral gavage. Tumors and terminal plasma were collected after 18 days of intervention for immunoblot and oxylipin analysis, respectively. There was no significant difference between treatment groups in body weights after 18 days, indicating a lack of general toxicity (Fig. 4A); tumor weights (Fig. 4B) and volume (Supplementary Fig. S1) were found to be the smallest in the mice treated with regorafenib while ingesting the DHA diet (∼1.9-fold decrease) and there was a synergistic decrease of the combination as compared with DHA or regorafenib administered alone.

To evaluate the target effects of regorafenib in the xenografted animals, we evaluated the MAPK and VEGFR pathways, which are known receptor tyrosine kinase targets (13). Immunoblotting of the tumors for pVEGFR-2 demonstrated the most dramatic reduction in the tumors from the DHA+regorafenib-treated mice with minimal effects upon these proteins in the other animals (Fig. 4C), indicating that regorafenib attenuates the active forms of both MAPK and VEGFR species, consistent with the HuVEC data (see Fig. 2C). Because we have previously shown an sEH inhibitory effect of regorafenib similar to sorafenib (45), the influence of regorafenib and DHA in the in vivo model is likely specific to this combination.

While the circulating plasma oxylipin profile can suggest the mechanism of the observations, these data do not always correlate with what is occurring at the local (i.e., tissue) level (46). The EDP species are rapidly metabolized to their diol constituents due to the actions of sEH; however, the inhibitory actions on this catabolic enzyme from an sEH-inhibitor, as we have shown for sorafenib (21, 47), were evident in the plasma (32). Terminal plasma oxylipin analysis showed the expected higher levels of 7(8)-EDP, 10(11)-EDP, 13(14)-EDP, 16(17)-EDP, and 19(20)-EDP in mice treated with the DHA diet compared with the corn oil diet groups (Fig. 5A). An increase in the corresponding diols was also observed as 10(11)-DiHDPE, 13(14)-DiHDPE, 16(17)-DiHDPE, and 19(20)-DiHDPE in the DHA-fed mice (Fig. 5B). The production of these diols was anticipated due to the enriched dietary DHA.

To assess in vivo sEH inhibition, we examined the ratio of epoxide to their corresponding diol products in the plasma. The sum epoxide-to-diol ratio was found to be ∼2.2-fold in the corn oil diet + regorafenib treatment group compared with the corn oil diet alone, with greatest difference being ∼3.6-fold increase found in the 16(17)-EDP-to-16(17)-DiHDPE (Fig. 5C). Surprisingly, the epoxide-to-diol ratio in the plasma of the DHA-fed mice did not reflect sEH inhibition as the concentrations were found to be about the same for all of the measured species, and even lower in the 10(11)-EDP-to-10(11)-DiHDPE and 13(14)-EDP-to-13(14)-DiHDPE. This was also observed in an earlier experiment performed with sorafenib rather than regorafenib treatments (data not shown). Recently, it has been identified that the omega-3–derived EDPs are turned over more rapidly than the corresponding omega-6–derived EETs as sEH has a preference for these DHA-derived epoxygenated metabolites (47). Thus, it is conceivable that due to this preference in substrate and the abundance of EDP in the blood, sEH enzyme levels may be upregulated and higher in the DHA-diet fed mice, resulting in a greater amount of epoxide turnover to diols, leading to a decrease in the epoxide-to-diol ratio in the plasma, although this may not be representative of tissue. Future investigations may elucidate this observation by measuring circulating EET and EDP concentrations or measuring oxylipins in other tissues.

We have shown that combination treatment of DHA with regorafenib results in a synergistic efficacy over regorafenib or DHA alone in inhibiting growth in an in vivo xenograft model of VHL-mut RCC. We further show that there is a decrease in markers of angiogenesis and that this growth inhibition is accompanied by the expected target effects. We provide evidence that tumor growth attenuation likely occurs as a result of increasing levels of EDPs due to the sEH inhibitory property of regorafenib (Fig. 6). Until a clinical trial is accomplished, patient use of the common and readily available dietary supplement, fish oil, can therefore be recommended in individuals undergoing regorafenib treatment for advanced RCC.

No potential conflicts of interest were disclosed.

Conception and design: J. Kim, A. Ulu, B.D. Hammock, R.H. Weiss

Development of methodology: J. Kim, A. Ulu, B.D. Hammock

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): J. Kim, A. Ulu, J. Yang

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): J. Kim, A. Ulu, D. Wan, J. Yang, R.H. Weiss

Writing, review, and/or revision of the manuscript: J. Kim, A. Ulu, D. Wan, J. Yang, B.D. Hammock, R.H. Weiss

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): J. Kim, D. Wan, B.D. Hammock, R.H. Weiss

Study supervision: J. Kim, R.H. Weiss

Other (worked with Dr. Weiss to develop original hypothesis based on previously published collaborative work): B.D. Hammock

This work was supported by NIH grants 1R01CA135401-01A1, 1R03CA181837-01, and 1R01DK082690-01A1, the Medical Service of the US Department of Veterans' Affairs, and Dialysis Clinics, Inc. (DCI; all to R.H. Weiss). Partial support was provided by NIEHS R01 ES002710 and NIEHS Superfund Program P42 ES004699 (to B.D. Hammock.)

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