The therapeutic value of SC66 in human renal cell carcinoma cells

The PI3K-AKT-mTOR cascade is required for renal cell carcinoma (RCC) progression. SC66 is novel AKT inhibitor. We found that SC66 inhibited viability, proliferation, migration and invasion of RCC cell lines (786-O and A498) and patient-derived primary RCC cells. Although SC66blocked AKT-mTORC1/2 activation in RCC cells, it remained cytotoxic in AKT-inhibited/-silenced RCC cells. In RCC cells, SC66 cytotoxicity appears to occur via reactive oxygen species (ROS) production, sphingosine kinase 1inhibition, ceramide accumulation and JNK activation, independent of AKT inhibition. The ROS scavenger N-acetylcysteine, the JNK inhibitor (JNKi) and the anti-ceramide sphingolipid sphingosine-1-phosphate all attenuated SC66-induced cytotoxicity in 786-O cells. In vivo, oral administration of SC66 potently inhibited subcutaneous 786-O xenograft growth in SCID mice. AKT-mTOR inhibition, SphK1 inhibition, ceramide accumulation and JNK activation were detected in SC66-treated 786-O xenograft tumors, indicating that SC66 inhibits RCC cell progression through AKT-dependent and AKT-independent mechanisms.


Introduction
Renal cell carcinoma (RCC) is the most common type of renal malignancy 1 . Nephroureterectomy of early-stage RCC is the only possible curable treatment option 1 . However, RCC is more often diagnosed at an advanced stage, with 25% of patients developing local invasion and systematic metastasis resulting in a poor prognosis 1 . The PI3K-AKT-mTOR signaling pathway is frequently upregulated in RCC due to mechanisms that include PTEN mutation/depletion, PI3KCA mutation, and sustained activation of receptor tyrosine kinases (RTKs) [2][3][4][5] . Constitutive activation of this cascade is necessary for RCC cell proliferation, survival, migration, and metastasis, and also angiogenesis and resistance to anti-tumor treatments 2,3,6,7 . Molecularly-targeted agents are currently being utilized for the treatment of certain RCC patients, including the mTORC1 inhibitors Temsirolimus and everolimus, which are approved by the FDA for the treatment of advanced RCC 2,3,6,7 .
Our group has previously shown that targeted inhibition of the PI3K-AKT-mTOR pathway is a valid treatment strategy in the management of RCC [8][9][10] . SF2523, a PI3K-AKT and bromodomain-containing protein 4 (BRD4) dual inhibitor, was found to potently inhibit RCC cell growth in vitro and in vivo 8 . Similarly, the AKT-mTORC1/2 inhibitor WYE-687 inhibited cell growth of human RCC cells 9 . In addition, we identified that microRNA-302c inhibited RCC cell proliferation by targeting Grb2-associated binding 2 (Gab2)-AKT signaling 10 .
Jo et al., developed SC66 11 , a novel allosteric AKT inhibitor that exerted a dual-inhibitory mechanism by inducing AKT ubiquitination and interfering with AKT pleckstrin homology (PH) domain binding to PIP3 11 . The study by Cusimano et al., demonstrated that AKT inhibition by SC66 induced significant cytotoxic effects in hepatocellular carcinoma (HCC) cells 12 . In this study, we demonstrate that in addition to AKT-dependent RCC cell inhibition, SC66 inhibits RCC cell progression via AKTindependent mechanisms.

Cell culture
The established RCC cells (786-O and A489 lines) and immortalized HK-2 tubule epithelial cells 13,14 were cultured using the previous protocol 10,15 . Cells were routinely subjected to mycoplasma and microbial contamination examination. STR profiling, population doubling time, and morphology were routinely checked every 3-4 months to confirm the genotype. The primary human RCC cells, derived from three primary RCC patients ("RCC1/2/3"), as well as the primary human renal epithelial cells ("Ren-Epi") were cultured in the described medium 8,9 . The written-informed consent was obtained from each enrolled patient. All investigations were conducted according to the principles expressed in the Declaration of Helsinki. Experiments and protocols were approved by the Ethics Review Board of Soochow University (Suzhou, China).

Methylthiazol tetrazolium (MTT) assay
Cells were seeded onto the 96-well tissue culture plates (3 × 10 3 cells per well). Following treatment, cell viability was assessed by the MTT assay. MTT OD was recorded at 490 nm.

Soft agar colony formation assay
Cells were seeded onto the 10-cm tissue culture dishes (1 × 10 4 cells per dish), treated with SC66 every two days for five rounds. Afterwards, the number of viable 786-O colonies were counted.

BrdU assay
Cells were seeded onto the six-well tissue culture plates (1 × 10 5 cells per well). Following treatment, cells were incubated with BrdU (10 μM, Cell Signaling Tech) for 8 h and then fixed. BrdU incorporation was determined in the ELISA format. BrdU OD at 405 nm was recorded.

EdU assay of cell proliferation
Cells were seeded onto the six-well tissue culture plates (1 × 10 5 cells per well). The EdU (5-ethynyl-20-deoxyuridine) Apollo-488 In Vitro Imaging Kit (Ribo-Bio, Guangzhou, China) was utilized to quantify cell proliferation. Following treatment EdU (2.5 μM) was added to RCC/epithelial cells for 6 h. Cell nuclei were stained with Hoechst-33342 for 5 min, visualized under a fluorescent microscope (Leica). We counted at least 400 cells of six random views to calculate EdU ratio for each treatment.
In vitro cell migration and invasion assays As described 16,17 RCC cells (4 × 10 4 cells of each condition in 200 μL serum-free medium) were initially seeded onto the upper surfaces of "Transwell" chambers. The lower compartments were always filled with complete medium (containing 10% FBS). Following 24 h incubation, the migrated cells on the lower surface were fixed, stained and counted. Matrigel (Sigma) was added in the chamber surfaces when analyzing cell invasion.

Caspase activity assay
Assaying of caspase-3/-9 activity was described previously 18 . Twenty μg of cytosolic extracts of each treatment were added to the caspase assay buffer 18 with the caspase-3 substrate or the caspase-9 substrate 18 . Release of 7-amido-4-(trifluoromethyl)-coumarin (AFC) was quantified by using a Fluoroskan system 18 . AFC optic density (OD) was recorded.

Annexin V FACS assay
As reported 18 , cells with the indicated treatment were washed and incubated with Annexin V-FITC (10 μg/mL) and propidium iodide (PI, 10 μg/mL) (Invitrogen), and detected by fluorescence-activated cell sorting (FACS) using a Becton-Dickinson machine. Annexin V-positive cells were labeled as the apoptotic cells.

TUNEL assay
Cells were seeded onto the six-well tissue culture plates (1 × 10 5 cells per well). Following treatment, cells were incubated with TUNEL (Invitrogen, 10 μM) for 3 h. Cell nuclei were stained with Hoechst-33342 for 5 min, visualized under a fluorescent microscope (Leica). For each treatment, we counted at least 400 cells of six random views (1×100 magnification) to calculate TUNEL ratio.

Western blotting assay
Cells and tumor tissues were incubated with RIPA lysis buffer (Biyuntian). Thirty micrograms of lysates per lane were separated on 10% sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis (PAGE) gels, and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore). After blocking, the blots were incubated with the applied primary and secondary antibodies. The enhanced chemiluminescence (ECL) reagents (GE Healthcare) were added to the blots to detect the targeted protein bands. Quantification of the band intensity was performed with Quantity One 4.6.2 software (Bio-Rad, Hercules, CA).

Reactive oxygen species (ROS) assay
As described 19 , the ROS levels were tested by using the carboxy-H2DCFDA dye. Following treatment, cells were stained with carboxy-H2-DCFDA (10 μM) for 30 min under the dark. The DCF fluorescence was measured under 485 nm excitation and 525 nm emission using the Fluorescence machine (Thermo Scientific, Shanghai, China).

Glutathione content assay
Reduced glutathione (GSH) is one key scavenger of ROS, and its ratio with oxidized disulfide form glutathione (GSSG) can be used as a quantitative indicator of oxidative stress intensity 20 . Following treatment, cells were lysed. The ratio of reduced to oxidized glutathione (GSH/ GSSG) was measured using the GSH/GSSG assay kit (Beyotime).

Ceramide content assay
The cellular ceramide level was analyzed by the protocol reported early 22 , tested as fmol by nmol of phospholipids.

AKT1 short hairpin RNA (shRNA)
AKT1 shRNA lentivirus (sc-29195V, Santa Cruz Biotech, 10 μL/mL medium) was added to 786-O cells for 24 h. Stable cells were selected by puromycin (5.0 μg/mL) for another 10 days. Expression of AKT1 in the stable cells was determined by Western blotting assay.

AKT knockout
The small guide RNA (sgRNA) targeting human AKT1 (Target DNA sequence, 5'-TCACGTTGGTCCA-CATCCTG) was inserted into the lenti-CRISPR-GFPpuro plasmid 25 . The construct was then transfected to 786-O cells by Lipofectamine 2000. FACS was performed to sort the GFP-positive 786-O cells. The resulting single cells were further cultured in the selection medium with puromycin (5 μg/mL) for 10 days. AKT1 knockout in stable cells was verified by Western blotting assay.

Xenograft model
Female CB-17 severe combined immunodeficiency disease (SCID) mice, 4-5 week old, 17-18 g, were provided by the Animal Center of Soochow University (Suzhou, China). 786-O cells (6 × 10 6 per mouse, in 200 μL DMEM/ Matrigel, no serum) were subcutaneously (s.c.) injected into flanks. After three week, the xenografts, close to 100 mm 3 , were established ("Day-0"). Ten mice per group were treated once daily by gavage with either vehicle control or SC66 (10 or 25 mg/kg body weight) for 24 consecutive days. Every six days, the mice body weights and bi-dimensional tumor measurements 18 were recorded. The animal protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of Soochow University and Ethics Review Board of Soochow University (Suzhou, China).

Statistical analysis
The investigators were blinded to the group allocation during all experiments. Results were expressed as the mean ± standard deviation (SD). Statistical analysis among different groups was performed via one-way analysis of variance (ANOVA) with Scheffe's test using SPSS20.0 software (SPSS Inc., Chicago, IL). The two-tailed unpaired T test (Excel 2007) was applied to test the significance of the difference between two treatment groups. P values of <0.05 were considered statistically significant.

SC66 inhibits 786-O xenograft tumor growth in SCID mice
We tested the potential effect of SC66 in vivo using the previously-described 786-O xenograft tumor model 8,9 . 786-O cells were s.c. injected into the flanks of SCID mice and xenografts established within three weeks when , or the primary human renal epithelial cells ("Ren_Epi") (g) were treated with indicated concentration of SC66, cells were further cultured for applied time periods, caspase-3/-9 activities (a), expression of apoptosis-associated proteins (b) and cell apoptosis (c-d, f, g) were tested by the mentioned assays. For e, 786-O cells were co-treated with 50 μM of the caspase-3 inhibitor z-DEVD-cho or the pan caspase inhibitor z-VAD-cho, and cell viability was tested by MTT assay. Expression of listed proteins were quantified, normalize to Tubulin (b). For each assay, n = 5. Data were expressed as the mean ± standard deviation (S.D.). *P < 0.05 vs. "Veh" group. # P < 0.05 vs. SC66 treatment only (e). In this figure, experiments were repeated three times, and similar results were obtained each time. Bar = 100 μm (d and f). (each at 5 μM), cells were further cultured for 72 h, and cell viability and apoptosis tested by MTT (g) and nuclear TUNEL staining (h) assays, respectively. Data were expressed as the mean ± standard deviation (S.D.). "DMSO" stands for 0.1% DMSO (g, h). *P < 0.05 vs. "Veh" group. # P < 0.05 vs . "DMSO" plus SC66 treatment (g, h). In this figure, experiments were repeated three times, and similar results were obtained each time.  a, 72 h). Stable 786-O cells with AKT1 shRNA ("sh-AKT1") or CRISPR/Cas9 AKT1-KO construct ("ko-AKT1"), as well as the control cells with scramble control shRNA and Cas9 empty plasmid ("scr-shRNA+ Cas9-c"), were tested by Western blotting assay of AKT expression (b, the upper panel), cells were treated with/without SC66 (3 μM) for 72 h, cell viability was tested (b, the lower panel). 786-O cells or the primary human RCC cells ("RCC1") were treated with SC66 (3 μM) for indicated time periods, ROS production (c and k), GSH/GSSG ratio (d), mitochondrial depolarization (e and l), SphK1 expression and activity (f), as well as the ceramide contents (g and m) and p-/t-JNK expression (h and n) were tested by the appropriate assays. 786-O cells were pretreated for 30 min with NAC (400 μM), JNKi (10 μM), or S1P (10 μM), followed by SC66 (3 μM) treatment for 48 and 72 h, cell viability and apoptosis were tested by MTT assay (h) and TUNEL staining assay (i), respectively. Phosphorylated JNK1/2 was normalized to total JNK1/2 (h and n). For each assay, n = 5. Data were expressed as the mean ± standard deviation (S.D.). *P < 0.05 vs. "Veh" group. # P < 0.05 vs . SC66 treatment only (i, j). In this figure, experiments were repeated three times, and similar results were obtained each time.
tumors were around 100 mm 3 ("Day-0"). We found that oral administration of SC66, at 10 and 25 mg/kg body weight, significantly inhibited tumor volume (Fig. 5a), and that daily tumor growth was significantly inhibited (Fig.  5b). At Day-36, tumors from all three groups were isolated and weighted individually.SC66-treated 786-O tumors weighted significantly less than the vehicle control tumors (Fig. 5c), while mouse body weights were not significantly different between the three groups (Fig. 5d). At treatment Day-6, two hours after SC66 (25 mg/kg) or vehicle administration, three 786-O tumors from each group (total six tumors) were isolated. Analyzing signaling changes, AKT-S6K phosphorylation was significantly inhibited in SC66-treated tumor lysates (Fig. 5e), confirming AKT-mTOR inhibition. In line with the in vitro findings, SC66 treatment decreased SphK1 activity (Fig. 5f), increased ceramide levels (Fig. 5g), and increased JNK activation (Fig. 5h).

Discussion
Our study shows that SC66 inhibited cell viability, proliferation, migration and invasion in established (786-O and A498 lines) and primary human RCC cells.SC66 was found to inhibit AKT-mTORC1/2 activation and induce significant apoptosis in RCC cells. In contrast, this AKT inhibitor was non-cytotoxic to HK-2 epithelial cells and primary human renal epithelial cells with low basal AKT-mTORC1/2 activation. In vivo, SC66 oral administration, at well-tolerated doses, potently inhibited subcutaneous 786-O xenograft growth in SCID mice. were recorded every 6 days. Estimated daily tumor growth was calculated as described (b). At Day-36, tumors were isolated and weighted (c). At treatment Day-6, two hours after SC66 (25 mg/kg) or vehicle administration, three 786-O tumors (n = 3) of each group (total six tumors) were isolated; Expression of listed proteins in tumor lysates was tested by Western blotting assays (e and h); The relative SphK1 activity (f) and ceramide contents (g) in tumor lysates were tested as well. The quantified results integrating all three sets of blotting data were presented (e and h). Data were expressed as the mean ± standard deviation (S.D.). *P < 0.05 vs. "Vehicle" group. mTORC1 inhibitors are approved by FDA for the treatment of advanced RCC patients after failure of either sunitinib or sorafenib 2,3 . However, the use of mTORC1 inhibitors can have several drawbacks. First, the mTORC1 inhibitors, rapamycin and its analogs ("rapalogs"), only indirectly inhibit mTORC1 38,39 . Second, mTORC1 inhibition can induce feedback activation of the PI3K-AKT and ERK-MAPK oncogenic pathways [38][39][40] . Third, rapalogs are unable to directly inhibit mTORC2, the latter being equally as important as mTORC1 in RCC progression. We have previously shown that WYE-687, a mTORC1/2 dual inhibitor, inhibited RCC cell growth with greater efficiency than mTORC1 inhibitors 9 . Further, the mTORC1/2 inhibitor, AZD2014, exerted more potent anti-RCC cell activity than rapalogs 18 . Similarly, the finding that SC66 can block AKT and mTORC1/2 activation in established and primary RCC cells is an advantage of this compound.
Furthermore, SC66 also exhibits cytotoxic actions independent of AKT1. Here, we show that in RCC cells SC66 induced ROS production, SphK1 inhibition, ceramide accumulation and JNK activation, which does not occur in RCC cells treated with the AKT specific inhibitor MK-2206 35,41 or in AKT1-silenced/-KO RCC cells. Significantly, the ROS scavenger NAC, the JNK inhibitor and anti-ceramide sphingolipid S1P all mitigated, but did not reverse, SC66-induced cytotoxicity in RCC cells. Importantly, confirming in vitro results, SphK1 inhibition, ceramide accumulation and JNK activation were detected in SC66-treated 786-O xenograft tumors. Therefore, SC66 acts through both AKT-dependent and AKT-independent mechanisms to exert more potent anti-RCC activity.
SphK1 is over-expressed and/or hyper-activated in RCC, promoting cancer progression 42,43 . SphK1 phosphorylates sphingosine to form S1P 44,45 , and SphK1 inhibition or silencing induces ceramide accumulation to promote cell apoptosis. Despite the importance of sphingolipid-derived signaling in tumorigenesis, there is a lack of potent and selective inhibitors of SphK. We found that SC66 inhibits SphK1 activation leading to proapoptotic ceramide accumulation and JNK activation in vitro and in vivo. Further studies are needed to determine the mechanism by which SC66 inhibits SphK1 in RCC cells.
It has been shown that Erk activation contributes to everolimus-acquired resistance and a poor prognosis in RCC patients 33 . Contrarily, Erk inhibition enhanced the efficacy of everolimus against RCC cells 33 . Yuen et al., found that AZD6244, an Erk inhibitor, at low doses augmented the antitumor activity of sorafenib 34 . In this study, we show that inhibition of Erk by PD98059 or U0126 potentiated SC66-induced cytotoxicity and apoptosis in 786-O and primary RCC cells, indicating that Erk activation could be a key resistance mechanism of SC66 in RCC cells.

Conclusion
In summary, we show that SC66 inhibits RCC cell progression in vitro and in vivo, through AKT-dependent and AKT-independent mechanisms. It should be noted that the findings of in vitro and animal RCC studies could not be directly translated to humans, and thus the efficacy and safety of SC66 need to be further investigated and confirmed.