Over-expression of DNA-PKcs in renal cell carcinoma regulates mTORC2 activation, HIF-2α expression and cell proliferation

Here, we demonstrated that DNA-PKcs is over-expressed in multiple human renal cell carcinoma (RCC) tissues and in primary/established human RCCs. Pharmacological or genetic inhibition of DNA-PKcs suppressed proliferation of RCC cells. DNA-PKcs was in the complex of mTOR and SIN1, mediating mTORC2 activation and HIF-2α expression in RCC cells. Inhibiting or silencing DNA-PKcs suppressed AKT Ser-473 phosphorylation and HIF-2α expression. In vivo, DNA-PKcs knockdown or oral administration of the DNA-PKcs inhibitor NU-7441 inhibited AKT Ser-473 phosphorylation, HIF-2α expression and 786-0 RCC xenograft growth in nude mice. We showed that miRNA-101 level was decreased in RCC tissues/cells, which could be responsible for DNA-PKcs overexpression and DNA-PKcs mediated oncogenic actions in RCC cells. We show that DNA-PKcs over-expression regulates mTORC2-AKT activation, HIF-2α expression and RCC cell proliferation.

RCC tissues, DNA-PKcs expression in RCC tissues was about 4-times higher than that in normal renal tissues (Fig. 1B). Real-time PCR assay results showed that DNA-PKcs mRNA level was also increased in RCC tissues (Fig. 1C).
Expression of DNA-PKcs in human RCC cells was also analyzed. As shown in Fig. 1D,E, DNA-PKcs protein expression was significantly higher in established (A498 and 786-0 lines) 20 and primary human RCC cells than that in non-cancerous proximal tubule epithelial HK-2 cells 20,21 . In addition, DNA-PKcs mRNA level was over-expressed in above HCC cells (Fig. 1F). Thus, these results show that DNA-PKcs is over-expressed in human RCC tissues and RCC cells.

DNA-PKcs inhibitors induce proliferation inhibition and apoptosis in RCC cells. Above results
demonstrate that DNA-PKcs is over-expressed in human RCC cells and RCC tissues. Next, we studied the potential effect of DNA-PKcs in RCC cell proliferation. Three different DNA-PKcs inhibitors, including NU-7026 22 , NU-7441 23 and LY-294002 24 were applied. Simply through the viable cell (trypan blue exclusive) counting assay, our results showed that the DNA-PKcs inhibitors remarkably inhibited 786-0 RCC cell proliferation ( Fig. 2A). Meanwhile, the results of MTT viability assay (Fig. 2B) and clonogenicity assay (Fig. 2C) further confirmed the anti-proliferative activity by these DNA-PKcs inhibitors. We also noticed significant apoptosis activation in 786-0 cells after treatment of DNA-PKcs inhibitors, which was shown by ssDNA apoptosis ELISA assay (Fig. 2D) and caspase-3 activity assay (Fig. 2E).
These DNA-PKcs inhibitors were also anti-proliferative in A498 RCC cells (Fig. 2F), and in primary human RCC cells 20 (Fig. 2G). Apoptosis induction, evidenced by ssDNA ELISA OD increase (Fig. 2H), was also observed in the primary cancer cells after the DNA-PKcs inhibitor treatment. On the other hand, the proliferation of non-cancerous HK-2 cells (low DNA-PKcs expression, Fig. 1) 21 were not affected by the same DNA-PKcs inhibitor treatment (Fig. 2I). Note that expression of DNA-PKcs was not affected by these inhibitors in above cells (Data not shown). Together, these results demonstrate that DNA-PKcs inhibitors exert anti-proliferative and pro-apoptotic activities to cultured RCC cells.
For the primary human RCC cells, siRNA method was applied to transiently knockdown DNA-PKcs, and Western blot assay showed DNA-PKcs silence by the two targeted siRNAs (Fig. 3H, upper). Primary human RCC cell proliferation, tested by MTT assay, was also inhibited by the non-overlapping DNA-PKcs siRNAs (Fig. 3H, lower). The proliferation of proximal tubule epithelial HK-2 cells was not affected by DNA-PKcs siRNAs (Fig. 3I,J). Thus, these results show that DNA-PKcs knockdown inhibits RCC cell proliferation in vitro.
DNA-PKcs is in the complex of mTOR and SIN1, required for mTORC2 activation and HIF-2α expression. Recent studies have demonstrated a potential role of DNA-PKcs in mTORC2 activation 16,25,26 .
It has been shown that DNA-PKcs could form a complex with SIN1, a key component of mTORC2 27,28 , thus regulating AKT Ser-473 phosphorylation 16,25 . Thus, we examined the potential role of DNA-PKcs in AKT-mTOR for applied time, cell proliferation was analyzed by viable cell counting assay (A, for 786-0 cells), MTT assay (B,F,G,I) or clonogenicity assay (C, for 786-0 cells); Cell apoptosis was tested by the ssDNA ELISA assay (D,H) or the caspase-3 activity assay (E, for 786-0 cells). Experiments in this figure were repeated three times, and similar results were obtained. For each assay, n = 5. * p < 0.05 vs. "DMSO" group.
signaling activation in RCC tissues. First, using Co-IP assay, we noticed a physical interaction between DNA-PKcs, SIN1, Rictor and mTOR in multiple human RCC tissues, and in 786-0 RCC cells (Fig. 4A). Raptor, the mTORC1 component 29 , was not in the complex (Fig. 4A). Same IP method failed to detect a significant SIN1-DNA-PKcs association in the above normal renal tissues (Data not shown), possibly due to low expression of both proteins (Fig. 1). As shown in Fig. 4B, DNA-PKcs-shRNA knockdown significantly inhibited AKT Ser-473 phosphorylation in 786-0 cells. Yet AKT Thr-308 phosphorylation was almost unaffected. Meanwhile, expression of HIF-2α , a mTORC2-regulated gene 30 , was also downregulated with DNA-PKcs knockdown (Fig. 4B). The HIF-1α expression, which was mainly regulated by mTORC1 30 , was not changed (Fig. 4B).
In addition, DNA-PKcs inhibitors, including NU-7026, NU-7441 and LY-294002, dramatically inhibited AKT Ser-473 phosphorylation and HIF-2α expression in 786-0 cells (Fig. 4C). Since LY-294002 was also a PI3K-AKT-mTOR pan inhibitor 31 , it thus blocked AKT Thr-308 phosphorylation and downregulated HIF-1α expression in 786-0 cells (Fig. 4C). As expected, SIN1-shRNA expressing cells showed similar results as DNA-PKcs-shRNA cells, showing decreased AKT Ser-473 phosphorylation and HIF-2α expression (Fig. 4D). AKT Thr-308 phosphorylation and HIF-1α expression were again not affected by SIN1 shRNA knockdown (Fig. 4D). Note that above experiments were also repeated in A498 cells and primary human HCC cells, and similar results were obtained (Data not shown). Together, these results indicate that DNA-PKcs is in the complex of mTORC2, regulating AKT Ser-473 phosphorylation and HIF-2α expression in RCC cells. Results demonstrated that oral administration of a single dose of the DNA-PKcs inhibitor NU-7441 (10 mg/kg, daily for three weeks) resulted in a significant inhibition of 786-0 xenograft growth in nude mice (Fig. 5A). Meanwhile, the in vivo growth of stable 786-0 cells with DNA-PKcs shRNA was also slower than the cells expressing scramble control shRNA (Fig. 5A). The daily xenograft growth volume was significantly lower with NU-7441 administration or DNA-PKcs silencing (Fig. 5B). Note that mice body weights were not affected by NU-7441 treatment nor by DNA-PKcs silencing (Fig. 5C). We also did not notice any signs of apparent toxicities, such as diarrhea, fever, severe piloerection or a sudden weight loss (> 10%), in the tested animals (Data not shown).
The signaling changes in the above xenografted tumors were also analyzed. In line with the in vitro findings, Western blot analysis of 786-0 xenografts (week-2 and week-4 after initial treatment) showed that AKT Ser-473 phosphorylation and HIF-2α expression were both inhibited by NU-7441 administration or DNA-PKcs shRNA knockdown in vivo (Fig. 5D). DNA-PKcs band confirmed its silence in 786-0 xenografts expressing DNA-PKcs-shRNA-1 (last for at least 2 to 4 weeks, Fig. 5D). Immunohistochemistry (IHC) results further confirmed that NU-7441 administration or DNA-PKcs shRNA suppressed AKT Ser-473 phosphorylation in 786-0 xenografts (two weeks after initial treatment, Fig. 5E). Collectively, these results show that DNA-PKcs inhibition or silencing suppresses AKT Ser-473 phosphorylation, HIF-2α expression and 786-0 xenograft growth in vivo.

miR-101 downregulation correlates with DNA-PKcs overexpression in RCC. Above results have
shown that DNA-PKcs overexpression regulates mTORC2 activation, HIF-2α expression and RCC cell proliferation. Next, we studied the underlying mechanisms of DNA-PKcs overexpression by focusing on miRNA regulation. A recent study by Yan et al. showed that miR-101 targeted 3′ UTR of DNA-PKcs mRNA, leading to DNA-PKcs mRNA degradation 19 . We thus analyzed level of miR-101 in human RCC tissues and RCC cells. Real-time PCR assay results in (Fig. 6A) showed that miR-101 was significantly decreased in RCC tissues ("Tumor") than that in surrounding normal renal tissues ("Normal") (n = 10). In addition, miR-101 level was also lower in established (A498 and 786-0 lines) and primary human RCC cells, as compared to HK-2 cells (Fig. 6B).

Discussions
In the present study, our results indicate that DNA-PKcs might be a novel oncogene for the RCC. First, we demonstrate that DNA-PKcs is over-expressed in multiple human RCC tissues. Second, inhibition of DNA-PKcs, through pharmacological inhibitors or siRNA/shRNA knockdown, significantly reduced RCC cell proliferation in vitro and in vivo. Third, DNA-PKcs was found in the complex of mTORC2, and was required for AKT activation (Ser-473 phosphorylation) and HIF-2α expression in RCC cells. Thus, DNA-PKcs might be a valuable target for RCC intervention.
Overactivity of AKT is observed in many RCCs, which plays a vital role in cell survival, proliferation, migration, apoptosis-resistances and other cancerous behaviors 33,34 . Complete activation of AKT requires both Ser-473 and Thr-308 phosphorylations 33,34 . Studies have indicated a potential role of DNA-PKcs in regulating AKT activation. For example, Feng et al. showed that DNA-PKcs directly associates and activates AKT in the plasma membrane, causing a 10-fold enhancement of AKT activity 35 16 . Similarly, Xu and co-authors found that, upon low-dose X-ray irradiation (LDI), DNA-PKcs associates with mTORC2 to mediate AKT Ser 473 phosphorylation 25 . In the current study, we showed that DNA-PKcs formed a complex with mTOR and SIN1 in both human RCC tissues and RCC cells, and was required for mTORC2 activation (AKT Ser-473 phosphorylation) and HIF-2α expression. Inhibition or silencing of DNA-PKcs in RCC cells reduced AKT Ser-473 phosphorylation and HIF-2α expression. Thus, DNA-PKcs may regulate RCC cell proliferation through regulating mTORC2 signaling.
Scientific RepoRts | 6:29415 | DOI: 10.1038/srep29415 production and tumor angiogenesis 38 . Studies have shown that 50% or more sporadic RCCs have somatic mutations in pVHL 38 . Although the role HIF-1α in tumor progression and angiogenesis has been extensively studied, existing evidences indicated that HIF-2α is far more important than HIF-1α in the pathogenesis of RCC 20,39,40 . As a matter of fact, HIF-2α silencing was shown to inhibit the ability of pVHL-knockout RCC cells to form tumors in vivo 39 . Kondo and colleagues showed that pVHL-mediated tumor suppression is abolished with overexpression of HIF-2α , but not HIF-1α 40 .
In the current study, we showed that HIF-2α expression was inhibited with DNA-PKcs silencing or blockage. These results were not surprising, since translation of HIF-2α is solely dependent upon the activity of mTORC2 30 , and we showed that DNA-PKcs was required for mTORC2 activation in RCC cells. As a matter of fact, SIN1 shRNA knockdown similarly decreased HIF-2α expression in 786-0 cells. Notably, HIF-1α expression was not affected by DNA-PKcs or SIN1 knockdown. One reason could be that HIF-1α translation is controlled mainly by mTORC1 30 . Collectively, we suggest that DNA-PKcs is in the complex of mTORC2, regulating AKT Ser-473 phosphorylation and HIF-2α expression in RCC cells.
miRNA-mediated gene regulation plays a fundamental role in controlling gene expression at the post-transcriptional level 41 . These miRNAs are vital in modifying many key biologic processes of human cells, possibly via regulating expression of signaling molecules including growth factors, cytokines, transcription factors and other proteins (i.e. DNA-PKcs 19 ) 41,42 . In addition, recent studies have shown that at least half of the miRNAs are linked to human cancers, these miRNAs are either upregulated or downregulated in human cancer cells. Specifically, many oncogenes and tumor suppressor genes are virtually regulated by miRNAs 42 . DNA-PKcs is shown to be negatively regulated by miRNA-101 19 . In the current study, we showed that miR-101 level was significantly lower in human RCC tissues, and in established or primary RCC cells, which might be a reason for DNA-PKcs over-expression. Introduction of miR-101 in RCC cells downregulated DNA-PKcs expression, and inhibited AKT activation, HIF-2α expression and cell proliferation. Reversely, over-expression of antagomiR-101 downregulated miR-101, and further enhanced DNA-PKcs expression and RCC cell proliferation. These results indicate that miR-101 downregulation might be at least one key reason for DNA-PKcs overexpression in RCC cells.
In summary, our results demonstrate that DNA-PKcs over-expression in RCC cells regulates mTORC2-AKT activation, HIF-2α expression and RCC cell progression. DNA-PKcs might be a valuable target for RCC treatment.  20 . For all the cell lines, DNA fingerprinting and profiling were performed every 6 months to confirm the origin of the cell line, and to distinguish the cell line from cross-contamination. All cell lines were subjected to mycoplasma and microbial contamination examination. Population doubling time, colony forming efficiency, and morphology under phase contrast were also measured every 6 months under defined conditions to confirm the phenotype of cell line.

Methods
Human RCC tissues. Tissue specimens were obtained from ten RCC patients with total nephroureterectomy. All patients were administrated in the Second Affiliated Hospital of Nantong University. Each patient received no irradiation or chemotherapy prior to surgery. In each fresh-isolated specimen, tumor tissue and the surrounding normal renal tissue were separated and paired. Tissues were thoroughly washed in PBS with antibiotics and DTT (2.5 mM, Sigma), and then minced into small pieces, which were then maintained in DMEM plus 10% FBS and necessary antibiotics. Tissues were lysed and analyzed by Western blots and real-time PCR. All patients enrolled provided individual written-informed consent. Using human specimens in this study was approved by the Nantong University's Scientific Ethical Committee (Approve ID: 2013-002). The methods were carried out in accordance with the principles set out in the Declaration of Helsinki and the NIH Belmont Report.
Primary culture of human RCC cells. Part of the minced RCC tumor tissues were also subjected to collagenase I (Sigma, 0.05% w/v) digestion for 30 min. Afterwards, individual cells were pelleted and rinsed twice with DMEM, and then cultured in DMEM, supplied with 10% FBS, 2 mM glutamine, 1 mM pyruvate, 10 mM HEPES, 100 units/mL penicillin/streptomycin, 0.1 mg/mL gentamicin, and 2 g/liter fungizone. Primary RCC cells of passage 3-6 were utilized for experiments.
Single-stranded DNA analysis of apoptosis. The single-stranded DNA (ssDNA) Apoptosis ELISA Kit (Chemicon International, Temecula, CA) was utilized to quantify cell apoptosis. This assay was based on selective DNA denaturation in apoptotic cells by formamide, and detection of the denatured DNA by monoclonal antibody to single-stranded DNA. The detailed procedure was described in other studies 45,46 . Caspase-3 activity assay. Caspase-3 activity assay was described in our previous study 20 . Briefly, ten micrograms of cytosolic extracts per treatment were added to caspase assay buffer (312.5 mM HEPES, pH 7.5, 31.25% sucrose, 0.3125% CHAPS) and the caspase-3 substrate (Calbiochem, Darmstadt, Germany). The release of 7-amido-4-(trifluoromethyl)-coumarin (AFC) was quantified via a Fluoroskan system set to an excitation value of 355 nm and emission value of 525 nm.

Western blots.
As previously reported 20 , cells or minced tissues were lysed by the lysis buffer containing 10 mM Tris-HCl (pH 7.4), 200 mM NaCl, 1 mM EGTA, 1% Triton X-100, 1 mM phenylmethylsulfonylfluoride, 10 μ g/mL leupeptin, 10 μ g/mL aprotinin, 100 mM NaF and 200 μ M sodium orthovanadate. Aliquots of 30 μ g of protein samples were separated by electrophoresis in SDS-PAGE, transferred to the PVDF membrane and detected with the specific antibody. The immunoreactive proteins after incubation with appropriately labeled secondary antibody were detected with an enhanced chemiluminescence (ECL) detection kit (Amersham, Buckinghamshire, UK). Band intensity was quantified by ImageJ software (NIH) after normalization to the loading control.

Co-Immunoprecipitation (Co-IP).
As previously reported 20 , aliquots of 1000 μ g of protein samples in 1 mL of lysis buffer from each treatment were pre-cleared by incubation with 30 μ L of protein A/G Sepharose (Sigma) for 2 hours at 4 °C rotation. The pre-cleared samples were incubated with the specific anti-SIN1 antibody (1 μ g/mL) overnight at 4 °C rotation. 20-30 μ L of protein A/G Sepharose were added to the samples 2 hours at 4 °C rotation. The beads were washed and boiled, followed by Western blot assay.
MTT assay of cell proliferation. Cells were seeded onto 96-well plates (3,000 per well) and allowed to attach overnight. After treatment of cells, cell viability/proliferation was tested using MTT [3-(4,5-dimet hylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay (Sigma) according to the manufacturer's instructions. Absorbance was measured at 490 nm through a Microplate Reader. The OD value of treatment group was always normalized to that of the control group.
Clonogenic assay. 786-0 cells were plated onto 60 mm plates with 2000 cells per plate. After treatment of cells, surviving colonies were fixed, stained with coomassie blue, and manually counted.
shRNA and stable cell selection. For shRNA experiments, lentiviral particles were produced by constructing a lentiviral GV248 expression vector (Genechem, Shanghai, China) containing a puromycin resistance gene and either scramble control shRNA (5′ -AATTCTCCGAACGTGTCACGT) 47 , shRNAs to DNA-PKcs (5′ -GAACACTTGTACCAGTGTT, DNA-PKcs shRNA-1) 47 and (5′ -GATCGCACCTTACTCTGTT, DNA-PKcs shRNA-2) 48 . SIN1 shRNA lentiviral particles were purchased from Santa Cruz Biotech (Santa Cruz, CA). For infection, RCC cells were grown in 6-well culture plates in the presence of 2.0 μ g/mL polybrene (Sigma) to 60% confluence, lentiviral-shRNAs were added to the cells. Virus-containing medium was replaced with fresh medium after 12 hours. Stable clones were selected by puromycin (0.5 μ g/mL) for 10 days, expression of targeted protein in the resistant colonies was tested by Western blots or real-time PCR. antagomiR-101 expression vector as well as miR-control ("miR-C") and pSuper-puro-GFP vector were gifts from Dr. Lu's Lab at Nanjing Medical University 32 . Cells were seeded onto 6-well plates at 50% confluence with 2.0 μ g/mL polybrene (Sigma). After 24 hours, cells were transfected using Lipofectamine 2000 transfection reagent (Invitrogen, USA). Twelve hours later, transfection medium was replaced with 2 mL of complete medium. Puromycin (2.5 μ g/mL, Sigma) was then added to select stable cells (8-10 days). Cells were always tested for miR-101.
Xenograft model. As previously reported 20 , eight-week-old female, nude/beige mice were purchased from Nantong University Animal Laboratories. Approximately 5 × 10 6 786-0 cells were injected into mice right flanks, and tumors were allowed to reach 10 mm in maximal diameter. Mice were divided into four groups (n = 10 of each group): stable 786-0 cells with scramble control shRNA, stable 786-0 cells with DNA-PKcs shRNA (-1), NU-7441 oral administration or vehicle administration. NU-7441 was initially solubilized as a stock solution of 10 mg/mL in ethanol. Prior to gavage, NU-7441 was brought up to volume (0.2 mL) in PBS with 0.5% TWEEN 80 Scientific RepoRts | 6:29415 | DOI: 10.1038/srep29415 and 2.5% N,N-dimethylacetamide. Mice body weight and bi-dimensional tumor measurements were taken every 7 days. Tumor volume was estimated using the standard formula: (length × width 2 )/2. Mice (1 mice per group) were sacrificed 7 day or 14 days after initial treatment, and the primary tumors were excised for Western blot and IHC staining analysis. Tumor xenografts were stored in liquid nitrogen. All experimental protocols were approved by the Nantong University's Institutional Animal Care and Use Committee (IACUC, Approve ID: 2013-015) and Nantong University's Scientific Ethical Committee (Approve ID: 2013-002). The methods were carried out in accordance with Nantong University's IACUC regulations. Animal surgery and euthanasia using decapitation were performed under Hypnorm/Midazolam anesthesia, and all efforts were made to minimize suffering. Immunohistochemistry (IHC) staining. The IHC staining was performed on cryostat sections (4 μ m/section) of xenograft tumors according to the described methods 32 . The slides were incubated with the primary antibody (anti-AKT Ser-473, 1:50), and subsequently stained with horseradish peroxidase (HRP)-coupled secondary antibody (Santa Cruz). The slides were then visualized via peroxidase activity using 3-amino-9-ethyl-carbazol (AEC) method (Merck, Shanghai, China).

Statistical analyses.
All experiments were repeated at least three times, and similar results were obtained.
Data were expressed as mean ± standard deviation (SD). Statistical analyses were analyzed by one-way analysis of variance (ANOVA). Multiple comparisons were performed using Tukey's honestly significant difference procedure. A p value of < 0.05 was considered statistically significant.