PI3K-Akt-mTOR inhibition by GNE-477 inhibits renal cell carcinoma cell growth in vitro and in vivo

GNE-477 potently inhibits human RCC cell survival, proliferation, cell cycle progression, migration and invasion

First the primary human RCC cells (“RCC1” [8, 9]) were cultured in FBS-containing complete medium, and treated with GNE-477 at 1-100 nM. After further culture for 24-96h, the cell viability was tested by CCK-8 assays. As demonstrated, GNE-477, in a dose-dependent manner, efficiently decreased CCK-8 viability in RCC1 cells (Figure 1A). The dual PI3K-mTOR inhibitor also displayed a time-dependent response in inhibiting CCK-8 viability in RCC1 cells (Figure 1A). The CCK-8 OD reduction was significant at 48h after GNE-477 treatment (10-100 nM), that lasted for at least 96h (Figure 1A). The colony formation assay results, Figure 1B, show that the number of viable RCC1 colonies was significantly decreased following GNE-477 treatment (at 10-100 nM, for 10 days). Since in RCC1 cells 50 nM of GNE-477 resulted in potent cell viability reduction (Figure 1A) and colony formation inhibition (Figure 1B), this concentration was selected for further experiments.

Figure 1. GNE-477 potently inhibits human RCC cell progression in vitro. The primary human RCC cells (“RCC1/RCC2/RCC3”), HK-2 renal epithelial cells (“HK-2”) or the primary human renal epithelial cells (“Epi”) were treated with applied concentration of GNE-477 (1-100 nM) or the vehicle control (“Veh”, 0.1% DMSO), cells were further cultured for the designated time periods (24-96h); Cell viability (A, G), colony formation (B) and proliferation (C, H) as well as cell cycle progression (D), migration (E, I) and invasion (F) were tested by the assays mentioned in the text. Bars stand for mean ± standard deviation (S.D.). For each assay, n=5. ** p < 0.01 vs. “Veh” cells. Experiments in this figure were repeated five times, and similar results obtained. Scale bar= 100 μm (C, E, F).

To study cell proliferation, a nuclear EdU staining assay was performed. Results show that GNE-477 (50 nM, 48h) treatment robustly inhibited EdU incorporation (EdU/DAPI%) in RCC1 cells (Figure 1C). Analyzing cell cycle progression by FACS, we show that S phases were potently decreased in GNE-477-treated RCC1 cells (Figure 1D), where G1 phases were increased (Figure 1D). Further studies demonstrated that GNE-477 (50 nM, 24h) suppressed in vitro cell migration (Figure 1E) and invasion (Figure 1F), tested by “Transwell” (Figure 1E) and “Matrigel Transwell” (Figure 1F) assays, respectively. Notably, for cell migration/invasion assays, RCC1 cells were treated with GNE-477 (50 nM) for only 24h, when no significant viability reduction was detected (Figure 1A).

In the primary human RCC cells-derived from two other RCC patients, RCC2 and RCC3, GNE-477 (50 nM) stimulation potently inhibited cell viability (CCK-8 OD, Figure 1G), proliferation (nuclear EdU incorporation, Figure 1H) and migration (Figure 1I). In contrast, in HK-2 renal epithelial cells and primary human renal epithelial cells, the same GNE-477 (50 nM) treatment was completely ineffective and non-cytotoxic (Figure 1G–1I). These results show that GNE-477 specially and potently inhibited RCC cell viability, proliferation, cell cycle progression, migration and invasion in vitro.

GNE-477 induces apoptosis activation in primary human RCC cells

To test cell apoptosis in GNE-477-treated RCC cells, the caspase activities were examined. As demonstrated, following GNE-477 (50 nM, 36h) treatment in RCC1 cells the caspase-3 activity (Figure 2A) and the caspase-9 activity (Figure 2B) increased over 8-10 folds (vs. vehicle control treatment). Western blotting assay results, Figure 2C, demonstrated that the dual PI3K-mTOR inhibitor induced cleavages of caspase-3, caspase-9 and PARP (poly (ADP-ribose) polymerase) in RCC1 cells. Further studies show that mitochondria depolarization was detected in GNE-477-treated RCC1 cells, evidenced by an increase of JC-1 green fluorescence intensity (Figure 2D). Additionally, following GNE-477 treatment about 25% of all RCC1 cell nuclei were positive for TUNEL staining (Figure 2E), indicating apoptosis activation.

Figure 2. GNE-477 induces apoptosis activation in primary human RCC cells. The primary human RCC cells (“RCC1/RCC2/RCC3”), HK-2 renal epithelial cells (“HK-2”) or the primary human renal epithelial cells (“Epi”) were treated with GNE-477 (50 nM) or the vehicle control (“Veh”, 0.1% DMSO), cells were further cultured for designated time periods (24-48h), and cell apoptosis tested by the mentioned assays (A–E, H, I). Alternatively, RCC1 cells were pretreated for 1h with applied caspase inhibitors (each at 50 μM), followed by GNE-477 (50 nM) stimulation, cells were further cultured for 48-72h, with cell apoptosis and viability examined by nuclear TUNEL staining (F) and CCK-8 (G) assays, respectively. Bars stand for mean ± standard deviation (S.D.). For each assay, n=5. ** p < 0.01 vs. “Veh” cells (A, B, D, E, H, I). ^##p < 0.01 vs. “DMSO”-pretreated cells (F, G). Experiments in this figure were repeated five times, and similar results obtained. Scale bar= 200 μm (E).

To confirm that apoptosis is the primary cause of GNE-477-induced cytotoxicity in RCC1 cells, a set of different caspase inhibitors were utilized. As demonstrated, pretreatment with the caspase-3 inhibitor z-DEVD-fmk, the caspase-9 inhibitor z-LEHD-fmk, or the pan-caspase inhibitor z-VAD-fmk, potently ameliorated GNE-477-induced apoptosis activation (TUNEL assay) in RCC1 cells (Figure 2F). Consequently, GNE-477-induced cytotoxicity, evidenced by CCK-8 viability reduction, was largely inhibited (Figure 2G). In other primary human RCC cells (RCC2 and RCC3), treatment with GNE-477 (50 nM) led to robust caspase-3 activation (Figure 2H) and nuclear TUNEL ratio increase (Figure 2I), indicating apoptosis activation. On the contrary, the dual PI3K-mTOR inhibitor failed to provoke significant apoptosis in HK-2 cells and primary kidney epithelial cells (Figure 2H, 2I). Together, these results show that GNE-477 induced robust apoptosis activation in primary human RCC cells.

GNE-477 blocks PI3K-Akt-mTOR cascade activation in primary human RCC cells

Next, the potential effect of GNE-477 on PI3K-Akt-mTOR signaling cascade was tested. As shown, in primary RCC1 cells treatment with GNE-477 (50 nM, 12h) almost completely blocked phosphorylation of p85 and Akt (Ser473 and Thr308) (Figure 3A). Phosphorylation of p70S6K1 and S6, the indicator of mTORC1 activation [15, 16], was largely inhibited in GNE-477-treated RCC1 cells as well (Figure 3A). Expression of total p85, Akt1, p70S6K1 and S6 was unchanged following GNE-477 treatment (Figure 3A). Furthermore, Erk1/2 phosphorylation and total Erk1/2 expression were not altered by GNE-477 in RCC1 cells (Figure 3A). These results indicated that GNE-477 blocked the whole PI3K-Akt-mTOR cascade activation in RCC1 cells.

Figure 3. GNE-477 blocks PI3K-Akt-mTOR cascade activation in primary human RCC cells. RCC1 cells were treated with GNE-477 (50 nM) or the vehicle control (“Veh”, 0.1% DMSO), cells were further cultured for 12h, and expression of listed proteins tested by Western blotting (A); RCC1 cells were treated with GNE-477 (“GNE”, 50 nM), LY294002 (100 nM), AZD2014 (“AZD”, 100 nM), perifosine (“Prf”, 1 μM) or the vehicle control (“Veh”, 0.1% DMSO) for 48-72h, with cell viability and apoptosis tested by CCK-8 (B) and nuclear TUNEL staining (C) assays, respectively. The monoclonal stable RCC1 cells with or without the constitutively-active Akt1 (“+ca-Akt1”) construct were treated with GNE-477 (50 nM) or the vehicle control, cells were further cultured for applied time periods, expression of the listed proteins was tested (D); Cell proliferation and apoptosis were tested by EdU staining (E) and TUNEL assay (F), respectively. The monoclonal stable RCC1 cells with the CRISPR/Cas9 Akt1-KO construct (Akt1-KO cells) were treated with or without GNE-477 (50 nM), control cells with empty vector were treated with vehicle control (“Veh”), expression of listed proteins was shown (G); Cell viability, proliferation and apoptosis were tested by CCK-8 (H), EdU incorporation (I), and TUNEL staining (J) assays after 48h, respectively. Expression of listed proteins was quantified, normalized to the loading control (A, D). Bars stand for mean ± standard deviation (S.D.). For each assay, n=5. ^##p < 0.01 vs. GNE-477 treatment (B, C). ** p < 0.01 (E, F). Experiments in this figure were repeated five times, and similar results obtained. Scale bar= 100 μm (E).

We next compared the anti-RCC cell activity of GNE-477 with other known PI3K-Akt-mTOR inhibitors, including a PI3K inhibitor LY294002 [17], an Akt specific inhibitor perifosine [18] and a mTOR kinase inhibitor AZD2014 [19]. In RCC1 cells, GNE-477-induced viability reduction and apoptosis (TUNEL ratio) were significantly more potent than those by LY294002, perifosine and AZD2014 (at even higher concentrations, Figure 3B, 3C).

Further studies demonstrated that a constitutively-active Akt1 (ca-Akt1) restored Akt and p70S6K1 phosphorylation in GNE-477-treated RCC1 cells (Figure 3D). Importantly, GNE-477-induced proliferation inhibition (EdU ratio decrease, Figure 3E) and cell apoptosis activation (nuclear TUNEL ratio increase, Figure 3F) were completely reversed by caAkt1 (Figure 3E, 3F). Therefore, forced activation of Akt reversed GNE-477-induced cytotoxicity in RCC1 cells, suggesting that PI3K-Akt-mTOR inhibition should be the cause of GNE-477-induced cytotoxicity against RCC cells. To further support our hypothesis, the CRISPR/Cas9 strategy was applied to complete knockout (KO) Akt1 in RCC1 cells (see Methods). Akt1 KO, mimicking GNE-477-induced activity, blocked Akt and p70S6K1 phosphorylation (Figure 3G), and led to robust viability reduction (Figure 3H), proliferation inhibition (Figure 3I) and cell apoptosis activation (Figure 3J). Importantly, in Akt1-KO RCC1 cells, adding GNE-477 (50 nM, 48h) was unable to induce further cytotoxicity (Figure 3H, 3I). These results further supported that GNE-477-induced cytotoxicity in RCC cells was due to PI3K-Akt-mTOR blockage.

GNE-477 potently inhibits RCC xenograft tumor growth in mice

The potential anti-RCC activity of GNE-477 in vivo was tested. Using a previously described animal model [8, 9], we subcutaneously (s.c.) injected RCC1 cells to the flanks of the nude mice. Within two weeks RCC1 xenograft tumors were established with the volume close to 100 mm³. Tumor growth curve results, Figure 4A, demonstrated that intraperitoneal (i.p.) injection of GNE-477, at 10 or 50 mg/kg (daily, for 3 weeks), potently inhibited RCC1 xenograft tumor growth in nude mice. Calculating the estimated daily tumor growth, using the formula (tumor volume at Day-35—tumor volume at Day-0)/35, we again show that GNE-477 injection potently suppressed RCC1 xenograft tumor growth in vivo (Figure 4B). At Day-35, tumors of all three groups were isolated and weighted. Tumors of GNE-477-treated mice were significantly lighter than those of the vehicle control mice (Figure 4C).

Figure 4. GNE-477 potently inhibits RCC xenograft tumor growth in mice. The nude mice bearing RCC1 xenograft tumors were intraperitoneally (i.p.) injected with GNE-477 (10-50 mg/kg body weight, daily, for 21 days), AZD2014 (10 mg/kg body weight, daily, for 21 days) or the vehicle control (“Veh”), tumor volumes (A) and mice body weights (D) were recorded every seven (7) days for a total of 35 days; Estimated daily tumor growth, in mm³ per day, was calculated (B); At the last day of recording (Day-35), the xenograft tumors were isolated and weighted (C). For each group, n=10 (mice). ** p < 0.01 vs. “Veh”-treated tumors (A–C). * p < 0.05 (A–C).

Notably, at the dose of 10 mg/kg (i.p. daily for three weeks) the known mTOR kinase inhibitor AZD2014 [19–21] significantly inhibited RCC1 xenograft tumor growth in mice (Figure 4A–4C). Importantly, GNE-477 was more potent in suppressing RCC1 xenograft growth than the same concentration of AZD2014 (Figure 4A–4C). There was no significant difference in animal body weights among the four groups (Figure 4D). Furthermore, no apparent toxicities were noticed in GNE-477-treated mice and AZD2014-treated mice. These results confirmed that GNE-477, at well-tolerated doses, potently inhibited RCC1 xenograft tumor growth in mice.

Chemicals and reagents

GNE-477 was synthesized by Min-de Biotech Co. (Suzhou, China) based on the published procedure [14]. LY294002, perifosine and AZD2014 were purchased from Selleck (Beijing, China). The caspase-3 inhibitor z-DEVD-fmk, the caspase-9 inhibitor z-LEHD-fmk, and the pan-caspase inhibitor z-VAD-fmk were obtained from RiboBio (Guangzhou, China). Antibodies of this study were obtained from Cell Signaling Tech (Beverly, MA) and Abcam Co. (Suzhou, China). All cell culture regents were from Gibco Co. (Shanghai, China). Puromycin, polybrene and JC-1 dye were provided by Sigma (St. Louis, MO).

Cell culture

As described previously [8, 9] the primary human RCC cells (from Dr. Zheng at Nantong University [32]) were derived from three independent primary RCC patients with written-informed consents. They were named as “RCC1”, “RCC2” and “RCC3”. The primary cancer cells were cultured in the previously-described medium for primary cells [33]. Cultures of HK-2 cells and primary human renal epithelial cells were also described in our previous study [9]. Protocols of this study were approved by the Ethics Board of Wenzhou Medical University, according to the principles expressed in the Declaration of Helsinki.

Cell viability

The primary human RCC cells or the epithelial cells were seeded onto 96-well plates (at 4, 000 cells per well). After the indicated treatments, the cell viability was examined by a Cell Counting Kit-8 (CCK-8) (Dojindo Molecular Technologies, Japan) CCK-8 optical densities (ODs) were examined using a microplate reader at the test-wavelength of 550 nm.

EdU (5-ethynyl-20-deoxyuridine) incorporation

The primary human RCC cells or the epithelial cells were seeded onto the six-well tissue-culturing plates (1×10⁵ cells per well). Following the indicated treatments an EdU Apollo-567 assay kit (RiboBio, Guangzhou, China) was utilized to test cell proliferation, with nuclear EdU and DAPI staining visualized under a fluorescent microscope (1×200 magnification, Leica, Shanghai, China.). In each treatment five random views with total 500 cells were included to calculate the nuclear EdU ratio (% vs. DAPI).

In vitro cell migration and invasion

The primary human RCC cells or the epithelial cells, with the applied treatments, were initially seeded on the upper chambers of “Transwell” (BD Biosciences, Shanghai, China) [34], at a density of 1×10⁴ cells in 250 μL serum-free medium in each chamber. The complete medium (15% FBS) was added to the lower chambers. After incubation for 24h, on the lower surface the migrated cells were stained and counted manually. To test cell invasion, Matrigel (Sigma, Shanghai, China) was added on the upper chambers of “Transwell”. For each condition, five repeated views were included to calculate the average number of migrated/invaded cells.

Cell cycle assay

The primary human RCC cells with or without GNC-477 treatment were stained with propidium iodide (PI, 5 μg/mL, Thermo-Fisher Invitrogen, Shanghai, China) and RNase (50 μg/mL, Thermo-Fisher Invitrogen). DNA contents were examined under a flow cytometer (BD Biosciences, Franklin Lakes, NJ). Cell cycle distributions were recorded.

Colony formation

RCC1 cells, in 0.25% agarose (Sigma)-containing complete medium, were initially seeded onto 10-cm tissue-culture dishes (at 1×10⁴ cells per dish). The FBS-containing complete medium with or without GNE-477 (at tested concentrations) was renewed every 2 days (for a total of 10 days). Afterwards, viable cell colonies were counted manually.

TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay

The protocols of nuclear TUNEL staining were described previously [8, 35]. Following the applied GNE-477 treatment, RCC cells were co-stained with TUNEL and DAPI. The TUNEL ratio (% vs. DAPI), calculating 500 nuclei per treatment in five random views (1:200), was recorded.

Western blotting

As described previously [8, 9], following the applied treatment, the quantified protein lysates (30-40 μg per sample) were separated by SDS-PAGE gels, that were transferred to the PVDF (polyvinylidene difluoride) blots (Merck-Millipore, Shanghai, China). After blocking (in milk-containing PBST), the blots were incubated with applied primary and secondary antibodies. The protein bands were visualized based on the molecular weights by using an enhanced chemiluminescence (ECL) kit (Pierce) [36]. Data quantification was through an ImageJ software (NIH, US).

Mitochondrial depolarization

As described [37], with mitochondrial depolarization in the stressed cells JC-1 red fluorescein shall aggregate into mitochondria to form green monomers [38]. Briefly, after the indicated treatments, RCC1 cells were stained with JC-1 dye at 10 μg/mL for 30 min under the dark. The JC-1 green intensity was examined via a fluorescence spectrofluorometer at 550 nm.

Constitutively-active mutant Akt1

The recombinant constitutively-active Akt1-GFP (caAkt1, S473D) adenovirus was provided by Dr. Zhang [39], that was transduced to RCC1 cells (cultured in the polybrene-containing complete medium). Cells were than subjected to FACS sorting of GFP to establish the monoclonal stable RCC1 cells, with caAkt1 expression verified by Western blotting analyses.

Akt1 knockout

A lenti-CRISPR/Cas9-GFP Akt1-KO construct was from Dr. Zhang’s lab at Soochow University [40], transduced to primary RCC1 cells. Cells were then subjected to FACS to sort GFP-positive cells, which were further distributed to 96-well tissue culture plates, with Akt1 knockout (KO) screened. The monoclonal stable Akt1 KO RCC1 cells were established.

Mice xenografts

As described previously [9], the female nude mice (5-6 week of age, 18.2-19.1 grams in weights) were provided by the Animal Center of Wenzhou Medical University (Wenzhou, China), maintained under standard conditions. Six million RCC1 cells per mouse were inoculated s.c. to the right flanks. Within two weeks the xenograft tumors were established with the volume close to 0.1 cm³. The tumor-bearing mice were randomly assigned into three groups (n=10 per group), intraperitoneally injected with GNE-477 or the vehicle control [14]. Tumor recordings were described early [8, 9]. The protocols were approved by the IACUC of Wenzhou Medical University, according to National Institutes of Health guide for the care and use of laboratory animals.

Statistical analyses

Data were presented as mean ± standard deviation (SD). Statistics were analyzed by one-way ANOVA using a SPSS software (21.0, SPSS Co., Chicago, CA). To test difference between two specific groups, a two tailed T Test was applied (Excel 2007, Microsoft). P values <0.05 were considered statistically different.