Three human renal cell carcinoma cell lines, UMRC6 (from B Zbar, National Cancer Institute, Bethesda, MD, United States), 786-0 (from W Kaelin, Dana Farber Cancer Institute, Boston, MA, United States), and UOK121 cells (from J. Gnarra, Louisiana State University, Baton Rouge, LA, United States) have been described previously (An et al., 2013). HEK-293 cells were purchased from Shanghai cell bank of the Chinese Academy of Sciences (Shanghai, China). Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Carlsbad, CA, United States) supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C in a humidified atmosphere of 5% CO₂. NVP-BEZ235 (Novartis Pharmaceuticals, Basel, Switzerland), PP242 (Sigma-Aldrich, St Louis, MO, United States), and Rapamycin (Santa Cruz Biotechnology, Santa Cruz, CA, United States) were solubilized in DMSO (in vitro assays) or a solution of 2% DMSO, 30% PEG 300, 5% Tween 80, and ddH2O (in vivo assays). Antibodies against Akt, phospho-Akt, TAK1, phospho-TAK1, IκB-α, phospho-IκB-α, c-Jun, and phospho-c-Jun were all purchased from Cell Signaling Technology (Beverly, MA, United States). Antibodies against phospho-mTOR, speckle-type POZ protein (SPOP), and β-actin were from Santa Cruz Biotechnology.

The two RCC tissue microarrays were purchased from Fanpu Biotech, Inc. (Guilin, Guangxi, China). One chip consisted of 87 patients with RCC (65 cases of clear cell RCC and 22 cases of papillary RCC) and samples of normal tissues from 8 patients. The other one included 60 patients with RCC (47 cases of clear cell RCC and 13 cases of papillary RCC) and samples of normal tissues from 5 patients. The chips were heated at 60°C to melt the paraffin, washed in xylene, and hydrated through graded ethanols (100–70%). For antigen retrieval, the chips were heated in Tris-EDTA for 20 min, and then blocked with 10% goat serum. Phospho-c-Jun and phospho-IκB-α staining was performed by incubation with rabbit anti-phospho-c-Jun antibody (1:100) and rabbit anti-phospho-IκB-α antibody (1:250) at 4°C overnight, followed by incubation of secondary antibody for 1 h at room temperature. Finally, the chips were stained with DAB chromogen solution and counterstained with hematoxylin.

The three RCC cell lines and HEK-293 cells were seeded in 96-well plates at a density of 1.5 × 10³ or 3.0 × 10³ per well. Cells were then treated with increasing doses (10, 100, 1,000 nM) of NVP-BEZ235, PP242, and Rapamycin for 48 h, or the equivalent amounts of DMSO as control. MTT (Sigma-Aldrich) was added to each well (1 mg/ml final concentration) and incubated at 37°C for 4 h. Then the plates were incubated with 100 μL of 10% SDS/0.01 N HCl. After incubation overnight, absorbance at 490 nM for each well was determined using a microplate reader (Bio-Rad, Sunnyvale, CA, United States). For colony formation assay, 786-0 and UOK121 cells were seeded in six-well plates with 500 cells per well, and cultured with 200 nM NVP-BEZ235, Rapamycin and PP242, or equivalent amounts of DMSO. Colonies were counted after 10–14 days of incubation.

Cell poptosis was analyzed using FITC Annexin V Apoptosis Detection Kit (BD Biosciences, San Jose, CA, United States). Briefly, after exposure to 100 nM of NVP-BEZ235, PP242, or Rapamycin for 72 h, cells were harvested, washed twice with cold PBS and resuspended in binding buffer at a density of 1 × 10⁶ cells/ml. About 1 × 10⁵ cells were stained with annexin V-FITC and propidium iodide (PI) for 15 min at room temperature in the dark and analyzed using a Beckman FC-500 flow cytometer (Beckman Coulter, CA, United States). For cell cycle assay, cells were respectively treated with different doses (100 and 500 nM) of NVP-BEZ235, PP242, and Rapamycin for 48 h, or equivalent amounts of DMSO. Then cells were fixed with 70% alcohol at 4°C overnight and stained with PI (5 ng/ml) for 20 min at room temperature. The cell distribution at various cell cycle phases were determined by flow cytometry.

First, the chamber membranes were coated using Matrigel (50 µg/ml). 2 × 10⁵ cells were added in the upper chambers with medium containing 1% FBS. The lower chamber was filled with DMEM containing 10% FBS with or without 200 nM of NVP-BEZ235, PP242, Rapamycin. After incubating for 48 h, cells invaded to the lower chamber were stained with 0.5% crystal violet, and counted using a microscope at 200 × magnification.

For time-dependent studies, cells were treated with 200 nM of NVP-BEZ235, PP242 and Rapamycin, or the equivalent amounts of DMSO as control. At different time points post-treatment, cells were harvested and lysed by incubation in lysis solution containing protease inhibitors. Equal amounts of total protein (20 or 40 µg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, United States). Membranes were probed with following primary antibodies: phospho- and total Akt, phospho-mTOR, SPOP, phospho- and total TAK1, phospho- and total c-Jun, phospho- and total IκB-α, β-actin. Band intensities were determined using ImageJ software (version 1.48; National Institutes of Health, Bethesda, MD, United States). For dose-dependent studies, cells were treated with increasing doses (10, 100, 500, 1,000 nM) of NVP-BEZ235, PP242, Rapamycin for 48 h. Protein was prepared and measured as described above. Data were expressed as ratio of phosphorylated component/total protein, p-mTOR/β-actin, or SPOP/β-actin.

For luciferase assay, RCC cell lines were transfected with the AP-1 or IκB-α reporter vector and a Renilla luciferase plasmid (Promega, Madison, WI, United States) as a control for transfection efficiency. To study whether AP-1 activation is regulated by TAK1/JNK pathway in RCC cells, the transfected cells were pretreated with TAK1-inhibitor 5Z-7-oxozeaenol (0, 50, 100, 200 nM) (Tocris Bioscience, Ellisville, MO, United States) or JNK-inhibitor II SP600125 (0, 2.5, 5, 10 µM) (Calbiochem, San Diego, CA, United States) for 1 h. Furthermore, to study the possible role of AP-1 and IκB-α in anticancer activities of NVP-BEZ235, PP242, and Rapamycin, the transfected cells were treated with these compounds (10, 100, 1,000 nM) for 24 h, or the equivalent amounts of DMSO as control. Luciferase activity was measured using a Dual-Glo^® Luciferase Assay System (Promega) according to the manufacturer’s protocol.

Four-week old female BALB/c nude mice were purchased from the Animal Experiment Center of Guilin Medical University (Guilin, Guangxi, China). 786-0 and A498 cells were chosen for in vivo study. A498 cells were purchased from Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, China). 1 × 10⁷ 786-0 or A498 cells were injected subcutaneously into nude mice. When the tumor xenografts reached around 300 mm³, mice were randomized into different groups (n = 6/group), and given NVP-BEZ235, PP242 or Rapamycin (15 mg/kg) every 2 days via oral gavage. Tumor size and mice weight were measured every other day. Mice were sacrificed after 28 days of treatment, and then the tumors were excised for histological examination. Tumor samples were fixed and embedded in paraffin. Then 5 μm tissues sections were deparaffinized and immunolabeled with antibodies against phospho-TAK1, phospho-IκB-α, and phospho-c-Jun. The intensity of staining versus background staining was visually determined under a light microscope. For the animal experiments, all procedures were approved by the Animal Research Ethics Committee of Guilin Medical University.

All data were expressed as mean ± standard deviation (SD). Statistical significance was determined using a one-way ANOVA followed by Tukey’s post hoc test using the SPSS statistical program (version 12.0; SPSS, Chicago, IL). A value of p < 0.05 was considered significant.

We here used three human renal cancer cell lines, UMRC6, 786-0, and UOK121, to access and compare the anticancer activities of NVP-BEZ235, PP242, and Rapamycin. It was found that the proliferation of all cell lines was significantly reduced by each compound (Figure 1A). Moreover, treatment with NVP-BEZ235 produced the greatest reduction in cell viability, followed by PP242 and Rapamycin. As control, the normal human embryonic kidney HEK-293 cells were also treated with NVP-BEZ235, PP242, and Rapamycin. Similar proliferation inhibition was induced by treatment with high concentrations of the three compounds, but not by 10 nM NVP-BEZ235 and PP242. It is indicated that at appropriate concentrations, NVP-BEZ235 and PP242 could exert anti-proliferative effects on RCC cells with less profound effect on normal cells. Likewise, the most marked decrease in numbers of colonies was observed in 786-0 and UOK121 cells treated with NVP-BEZ235 (Figure 1B).

Additional studies were done to address the antiproliferative mechanism of NVP-BEZ235, PP242, and Rapamycin. Flow cytometric analysis of the cell cycle distribution of RCC cells showed that the cells were partially blocked in the G1 phase in all cultures, especially in NVP-BEZ235-treated cell lines (Figure 2). Cells at the G1 phase increased from 54.54% in control group to 69.03% (500 nM NVP-BEZ235) in UMRC6 cells, from 54.76 to 71.79% in 786-0 cells, from 51.79 to 73.30% in UOK121 cells. Meanwhile, we observed that the percentage of apoptotic cells increased significantly in RCC cells when treated with the three compounds (Figure 3A). Also, NVP-BEZ235 possessed higher pro-apoptotic activity than PP242 and Rapamycin. These findings indicate that apoptosis induction and cell cycle arrest contribute to the growth inhibition of RCC cells by NVP-BEZ235, PP242, and Rapamycin. At the same time, these compounds significantly reduced cell migration in the order NVP-BEZ235 > PP242 > Rapamycin (Figure 3B).

NVP-BEZ235, PP242, and Rapamycin are inhibitors of PI3K/Akt/mTOR pathway. Consistent with previous studies, it was shown that NVP-BEZ235 and PP242 inhibited phosphorylation of Akt and mTOR in UMRC6, 786-0, and UOK121 cells, but Rapamycin only decreased p-mTOR expression in the three cell lines (Figures 4A,B; Supplementary Figure S1). On the contrary, Rapamycin induced feedback activation of Akt in UMRC6 cells, in coincidence with previous report (Osawa et al., 2019). Nevertheless, in UOK121 and 786-0 cells, Rapamycin produced no significant change in Akt phosphorylation, though there was an obvious decrease in p-Akt levels within the first 8 h after exposure in 786-0 cells. In view of the relationship between SPOP mutations and activation of PI3K pathway in cancers, we next detected the alteration of SPOP in RCC cells. NVP-BEZ235 and PP242 suppressed SPOP protein expression in RCC cell lines in a dose- and time-dependent manner, coincident with reduced Akt phosphorylation, whereas no obvious change was observed in Rapamycin-treated cells after 48 h of exposure (Figure 4C; Supplementary Figure S2A). Furthermore, in comparison with PP242, NVP-BEZ235 had a superior effect on reduction of SPOP expression and inactivation of PI3K/Akt/mTOR pathway, and this effect was more pronounced in 786-0 and UOK121 cells.

In present study, the elevated phosphorylation levels of c-Jun and IκB-α were found in RCC tissue samples, including both clear cell and papillary renal cell carcinomas, confirming the participation of JNK and IKK activation in the development and progression of renal carcinoma (Figure 5). Thus, we here focused on TAK1, a member of the mitogen-activated protein kinase kinase kinase (MAPKKK) family that can function in the JNK and IKK pathways (An et al., 2013). Exposure of RCC cell lines to NVP-BEZ235 and PP242 reduced phosphorylation of TAK1 in a time- and dose-dependent manner (Figure 6D). Additionally, the specific inhibitors of TAK1 and JNK both dose-dependently provoked a reduction of AP-1 activity, confirming that AP-1 acts as downstream of TAK1, and JNK in RCC cells (Figure 6A). Correspondingly, the inhibition on TAK1 and JNK by NVP-BEZ235 and PP242 was accompanied with reduced AP-1 activity and inhibition of c-Jun phosphorylation (Figures 6B,E; Supplementary Figures S2B, S3A). On the other hand, although Rapamycin suppressed AP-1 activation in 786-0 and UOK121 cells, it failed to significantly inhibit phosphorylation of TAK1 and c-Jun in RCC cell lines, except for a temporary down-regulation of p-TAK1 and p-c-Jun expression. IκB-α is another downstream molecule of TAK1, and its phosphorylation is mainly mediated by IKK. Subsequent to decreased TAK1 phosphorylation, the activation of IκB-α was steadily and significantly inhibited by NVP-BEZ235 and PP242 rather than Rapamycin in RCC cells (Figures 6C,F; Supplementary Figure S3B). The decline trend of c-Jun/AP-1 activity, Akt, and IκB-α phosphorylation was much more marked in RCC cells treated with NVP-BEZ235. These results demonstrated that TAK1-dependent JNK and IKK signaling is involved in anti-cancer mechanism of NVP-BEZ235 and PP242 in renal cell carcinoma, which may be a new possible explanation to the different response of RCC cells to these compounds.

To determine the in vivo efficacy of NVP-BEZ235, PP242 and Rapamycin against human renal cancer, nude mice bearing 786-0 or A498 tumor xenograft were treated daily with these compounds as described in Methods. As shown in Figure 7B, no significant change was observed in body weight in 786-0 xenograft model within each group, while the mice administrated with PP242 and Rapamycin showed decreased body weights as compared with the weights of the controls in A498 xenograft mouse model. In general, the tumors induced by 786-0 or A498 cells injection were both most sensitive to treatment with NVP-BEZ235, followed by PP242 (Figures 7A,C). At the end of the experiment, the tumor volumes of the nude mice bearing 786-0 and A498 cells were respectively reduced by 75 and 44.6% with treatment of NVP-BEZ235, while PP242 treatment reduced tumor volumes by only 31% in 786-0 xenografts and 10.5% in A498 xenografts. Instead, Rapamycin displayed modest anti-cancer properties in vivo or, unexpectedly, even slightly promoted tumor growth at the late stage in mice bearing A498 tumor xenograft. By immunohistochemical staining, we demonstrated that NVP-BEZ235, and PP242 caused decrease in phosphorylation of TAK1, c-Jun and IκB-α in tumor tissues, indicating that the in vivo anticancer activity of NVP-BEZ235 and PP242 against RCC also depends on TAK1/JNK and TAK1/IKK signaling pathways (Figure 8). In agreement with the in vitro results, these decrease was more pronounced in NVP-BEZ235-treated xenograft model, while Rapamycin showed no inhibitory effects on phosphorylation of these molecules.