KU-0060648 inhibits hepatocellular carcinoma cells through DNA-PKcs-dependent and DNA-PKcs-independent mechanisms

Here we tested anti-tumor activity of KU-0060648 in preclinical hepatocellular carcinoma (HCC) models. Our results demonstrated that KU-0060648 was anti-proliferative and pro-apoptotic in established (HepG2, Huh-7 and KYN-2 lines) and primary human HCC cells, but was non-cytotoxic to non-cancerous HL-7702 hepatocytes. DNA-PKcs (DNA-activated protein kinase catalytic subunit) is an important but not exclusive target of KU-0060648. DNA-PKcs knockdown or dominant negative mutation inhibited HCC cell proliferation. On the other hand, overexpression of wild-type DNA-PKcs enhanced HepG2 cell proliferation. Importantly, KU-0060648 was still cytotoxic to DNA-PKcs-silenced or -mutated HepG2 cells, although its activity in these cells was relatively weak. Further studies showed that KU-0060648 inhibited PI3K-AKT-mTOR activation, independent of DNA-PKcs. Introduction of constitutively-active AKT1 (CA-AKT1) restored AKT-mTOR activation after KU-0060648 treatment in HepG2 cells, and alleviated subsequent cytotoxicity. In vivo, intraperitoneal (i.p.) injection of KU-0060648 significantly inhibited HepG2 xenograft growth in nude mice. AKT-mTOR activation was also inhibited in xenografted tumors. Finally, we showed that DNA-PKcs expression was significantly upregulated in human HCC tissues. Yet miRNA-101, an anti-DNA-PKcs miRNA, was downregulated. Over-expression of miR-101 in HepG2 cells inhibited DNA-PKcs expression and cell proliferation. Together, these results indicate that KU-0060648 inhibits HCC cells through DNA-PKcs-dependent and -independent mechanisms.


KU-0060648 inhibits HCC cell proliferation
To test the potential role of KU-0060648 on HCC cells, HepG2 cells were treated with applied concentrations of KU-0060648. MTT assay results in Figure 1A demonstrated that KU-0060648 dose-dependently inhibited HepG2 cell proliferation, with IC50 = 134.32 ± 7.12 nM. Proliferation inhibition by KU-0060648 in HepG2 cells was also confirmed by results from the [H 3 ] Thymidine incorporation assay (Supplementary Figure  S1A). Meanwhile, KU-0060648 (at 300 nM) also showed a time-dependent effect in inhibiting HepG2 cells ( Figure  1B). Further, the clonogenicity assay results in Figure  1C again demonstrated the anti-proliferative activity by KU-0060648. The number of viable HepG2 colonies was significantly decreased following applied KU-0060648 (30-500 nM) treatment ( Figure 1C). Notably, KU-0060648 exerted similar anti-proliferative effect in two other human HCC cell lines: Huh-7 and KYN-2 ( Figure 1D and Supplementary Figure S1B). human HCC cells E. line-1/-2) and HL-7702 human hepatocytes F. were either left untreated ("Ctrl", same for all figures), or treated with applied concentrations of KU-0060648 ("KU", 30-500 nM), cells were then cultured for indicated time. Cell proliferation was tested by MTT assay (A and B, D-F) or clonogenicity assay (C). IC-50 was calculated by the SPSS software (A and D). Experiments in this figure were repeated four times, with similar results obtained. n=5 for each repeat. Bars stand for mean ± SD * p < 0.05 vs. group "Ctrl". www.impactjournals.com/oncotarget The potential activity of KU-0060648 in primary human HCC cells was also tested. Using the method described, we successfully cultured two primary human HCC cell lines. These cells were treated with KU-0060648. Results of MTT assay ( Figure 1E) Figure  S1B) demonstrated clearly that KU-0060648 inhibited primary HCC cell proliferation. Significantly, same KU-0060648 treatment was general safe to non-cancerous HL-7702 human hepatocytes ( Figure 1F). Only exception was KU-0060648 at 500 nM, which only slightly inhibited HL-7702 cell proliferation ( Figure 1F). One reason could be that HL-7702 hepatocytes express very low level of DNA-PKcs, as compared to primary HCC cells (Supplementary Figure S1C). Further, MTT assay results showed that KU-0060648 was mostly ineffective to the proliferation of two different types of non-cancerous cells, including the human peripheral blood mononuclear cells (PBMCs) and primary human skin fibroblasts (HSFs) (Supplementary Figure S1D). Note that these non-cancerous cells grew much slower than primary and established (HepG2) HCC cells (Supplementary Figure S1E). Together, these results indicate a selective and potent anti-proliferative activity by KU-0060648 against HCC cells.

KU-0060648 induces caspase-dependent HCC cell apoptotic death
The results above demonstrated that KU-0060648 exerted potent anti-proliferative activity against human HCC cells. We next wanted to know if apoptosis activation was occurred. Two independent assays, including the caspase-3 activity assay and the histone DNA apoptosis ELISA assay [21,24], were performed. Results from both assays showed that KU-0060648 at 100 and 300 nM induced significant apoptosis activation in HepG2 cells (Figure 2A and 2B). The caspase-3 activity and the apoptosis ELISA OD were both increased following KU-0060648 treatment (Figure 2A and 2B). The caspae-3 specific inhibitor z-DEVD-fmk and the general caspase inhibitor z-VAD-fmk largely inhibited KU-0060648induced apoptosis activation in HepG2 cells ( Figure  2A and 2B). Importantly, KU-0060648-induced anti-HepG2 cell activity, evidenced by MTT OD reduction, was significantly attenuated with pretreatment of the two caspase inhibitors ( Figure 2C).
Therefore, first we showed that DNA-PKcs inhibition (by adding NU-7441 and NU-7026), silence (by shRNAs), or mutation (T2609A) didn't reach to same degree of HepG2 cell inhibition as KU-0060648 did. Second, KU-0060648 was still anti-proliferative and pro-apoptotic in the DNA-PKcs-silenced/-mutated cells. Third, KU-0060648 could still inhibit HepG2 cells that were already treated with known DNA-PKcs inhibitors (NU-7441 and NU-7026). These results suggest that DNA-PKcs is first a vital molecule for HCC cell survival and proliferation. Second, it is an important yet not exclusive target of KU-0060648. DNA-PKcsindependent mechanisms should also play an important role in mediating KU-0060648's actions in HCC cells.
In primary human HCC cells, targeted-siRNA was applied to transiently knockdown DNA-PKcs ( Figure 3G). DNA-PKcs siRNA knockdown inhibited primary HCC cell growth ( Figure 3H). Similarly, KU-0060648 could still induce an anti-proliferative activity in DNA-PKcssilenced primary cancer cells ( Figure 3H). Based on above results, we would propose that DNA-PKcs overexpression could promote HCC cell proliferation. Using the method described, we established stable HepG2 cells expressing wild-type DNA-PKcs (wt-DNA-PKcs, Flag-tagged) ( Figure 3I). Western blotting assay confirmed DNA-PKcs overexpression in the stable HepG2 cells ( Figure 3I). Consequently, cell proliferation, tested by MTT assay ( Figure 3J) and cell number counting ( Figure 3K), was also enhanced. These results together indicate that DNA-PKcs is an important but not exclusive target of KU-0060648 in HCC cells.
Next, we tested whether PI3K-AKT-mTOR inhibition is a secondary effect of DNA-PKcs inhibition. We showed that activation of AKT (Ser-473 phosphorylation) and mTOR (S6K1 phosphorylation) was intact in DNA-PKcs-silenced ( Figure 4G) or DNA-PKcs-mutated ( Figure 4H) HepG2 cells. Further, primary HCC cells with DNA-PKcs siRNA also showed equivalent AKT-mTOR activation, as compared to scramble siRNAtransfected HCC cells ( Figure 4I). Meanwhile, AKT-mTOR activation was also unchanged in DNA-PKcs- Cell proliferation E. and apoptosis F. were also tested. Expressions of listed kinases in HCC cells described in Figure 3 were tested G-J. Kinase phosphorylation (vs. total kinase) was quantified. Experiments in this figure were repeated three times, with similar results obtained. n=5 for each repeat (E and F). Bars stand for mean ± SD *p < 0.05. overexpressed HepG2 cells ( Figure 4J). These results indicate that PI3K-AKT-mTOR inhibition is likely a direct action by KU-0060648 in HCC cells, independent of DNA-PKcs inhibition.

KU-0060648 suppresses HepG2 xenograft growth in nude mice
We also tested the in vivo activity of KU-0060648 using the HepG2 xenograft nude mice model. As described, a significant number of HepG2 cells were injected into the right flanks of nude mice, and xenografted tumors were established ( Figure 5A). Administration of KU-0060648 (i.p. 10 and 50 mg/kg bodyweight, daily for 21 days) [12] dramatically inhibited HepG2 xenograft growth in nude mice ( Figure 5A). Tumor daily growth in KU-0060648-admnistrated mice was significantly lower than that in vehicle control (saline) mice ( Figure 5B). Further, the tumor weights (at week-5) of KU-0060648 group mice were also dramatically lighter than that of vehicle control mice ( Figure 5C). Notably, KU-0060648 exerted a dose-dependent effect in vivo, KU-0060648 at 50 mg/kg was more potent than 10 mg/kg in inhibiting HepG2 xenografts ( Figure 5A-5C). As shown in Figure  5D, mice body weights were almost not changed between each group in the tested durations, indicating that KU-0060648 administrations was generally safe to the experimental mice. We also failed to notice any deleterious side-effects in tested animals (vomiting, diarrhea, sudden weight loss, fever etc). Thus, we show that KU-0060648, at well-tolerated doses, suppresses HepG2 xenograft growth in nude mice.
We also tested the effect of KU-0060648 on AKT-mTOR activation in vivo. First, Western blotting was utilized to analyze signaling changes in above HepG2 xenografts (two mice per set). Results in Figure 5E showed clearly that administration of KU-0060648 dramatically inhibited AKT-mTOR activation in HepG2 xenografts. High-dose (50 mg/kg) of KU-0060648 was again more potent than low-dose (10 mg/kg) in inhibiting AKT-mTOR activation ( Figure 5E). IHC staining results analyzing p-AKT Ser473 in Figure 5F (Set-1) further confirmed AKT inhibition by KU-0060648 administration, similar results were obtained in Set-2 (Data not shown). 10 for growth assay, 2 for signaling assay) were administrated with KU-0060648 (i.p. 10 and 50 mg/kg bodyweight, daily, for 21 days) or the vehicle control (saline), tumor volumes (in mm 3 ) A. and mice body weights (in gram) D. were presented weekly, tumor daily growth was also calculated B. At the end of the experiments (week-5), tumors were separated through surgery and weighted C. Two weeks after initial KU-0060648 administration, two mice per group were sacrificed, HepG2 tumor tissues were isolated for Western blotting assay E. or IHC staining assay F. of indicated proteins, representative p-AKT (Ser473) IHC images were presented (Bar=50 μm) (F). Kinase phosphorylation (vs. total kinase) was quantified (E). Experiments in this figure were repeated twice, with similar results obtained. Bars stand for mean ± SD. "w" stands for week. *p < 0.05 vs. group "Vehicle". # p < 0.05 vs. KU-0060648 at 10 mg/kg group.
Thus, in line with the in vitro findings, i.p. administration of KU-0060648 inhibits AKT-mTOR activation in HepG2 xenografts.

DNA-PKcs upregulation in human HCC cells and tissues, correlated with miRNA-101 downregulation
Finally, we tested DNA-PKcs expression in human HCC tissues ("Tumor tissues"), and compared its level with surrounding normal liver tissues ("Liver tissues"). As demonstrated in Figure 6A, DNA-PKcs protein was overexpressed in HCC tissues (derived from eight different HCC patients). Its expression level was significantly higher in "Tumor tissues" vs. surrounding "Liver tissues" ( Figure 6B). DNA-PKcs mRNA expression was also upregulated in HCC tissues ( Figure 6C). We next studied the possible cause of DNA-PKcs upregulation in HCC by focusing on microRNAs (miRs). miRs are capable of regulating gene expression at translational or posttranscriptional levels [34,35]. The 19-24 nucleotide singlestranded noncoding RNAs are shown to silence targeted mRNAs translation with partial complementarity in their 3′ untranslated regions (UTRs) [34,35]. Existing evidences have shown that miR-101 could direct bind to and sequester DNA-PKcs mRNA [36]. Results in Figure 6D demonstrated clearly that miR-101 level in HCC tissues was significantly lower than that in surrounding normal liver tissues, which might be responsible for DNA-PKcs mRNA/protein upregulation in HCCs ( Figure 6A and 6C). (Protein and mRNA) and miRNA-101 ("miR-101") expressions in surgery-isolated fresh human HCC tumor tissues ("Tumor tissues") and surrounding normal liver tissues ("Liver tissues") were shown A, C. and D. Protein expression of DNA-PKcs (vs. Tubulin) was quantified B. Stable HepG2 cells transfected with miR-101 construct, nonsense miRNA-control construct ("miR-C"), or the empty vector (pSuperpuro, "Vector"), were subjected to real-time PCR assay E-F. or Western blotting assay G. to test DNA-PKcs and miRNA-101 expressions. Proliferation H. and I. and apoptosis J. in above cells were also tested. Experiments in this figure were repeated three times, with similar results obtained. n=5 for each repeat. Bars stand for mean ± SD *p < 0.05. Figure S3B and S3C demonstrated low miR-101 expression yet high DNA-PKcs mRNA expression in established or primary HCC cells, as compared to the non-cancerous HL-7702 cells. When we exogenously overexpressed miR-101 in HepG2 cells ( Figure 6E), DNA-PKcs mRNA ( Figure 6F) and protein ( Figure 6G) expressions were correspondingly downregulated. As a result, HepG2 cell proliferation, tested by MTT assay ( Figure 6H) and cell counting assay ( Figure  6I), was inhibited. These miR-101-expressing HepG2 also showed spontaneous cell apoptosis ( Figure 6J). As expected, nonsense miRNA-control construct ("miR-C") showed no effect on DNA-PKcs expression or HepG2 cell proliferation ( Figure 6E-6I). These results demonstrate DNA-PKcs overexpression in human HCC tissues, which is correlated with miRNA-101 downregulation.

DISCUSSIONS AND CONCLUSIONS
The preclinical results of the current study indicate that KU-0060648, a novel DNA-PKcs inhibitor, could be a potential anti-HCC agent. First, expression of DNA-PKcs was upregulated in the tested human HCC tissues. Second, DNA-PKcs knockdown or mutation inhibited human HCC cell proliferation. Third, KU-0060648 exerted potent antiproliferative and pro-apoptotic activities in established and primary human HCC cells. It was yet non-cytotoxic to human HL-7702 hepatocytes. Fourth, KU-0060648 suppressed PI3K-AKT-mTOR activation in human HCC cells. Fifth, intraperitoneal (i.p.) injection of KU-0060648 dramatically inhibited HepG2 xenograft growth in nude mice without causing apparent toxicities. Thus, DNA-PKcs is an important oncotarget for HCC [36,37], and KU-0060648 might merit further investigations as a valuable anti-HCC agent.
We propose that the DNA-PKcs is an important but not exclusive target of KU-0060648 in HCC cells. HepG2 cells with DNA-PKcs silence (by shRNA/ siRNA) or mutation displayed decreased proliferation and spontaneous apoptosis. Significantly, KU-0060648 was still anti-proliferative and pro-apoptotic in DNA-PKcssilenced or -mutated cells, although its activity in these cells was relatively weak. Another important target by KU-0060648 in HCC cells could be PI3K-AKT-mTOR [12]. Overactivation of PI3K-AKT and its downstream mTORC1-S6K1 cascade is vital for HCC progression [38] [3, [39][40][41]. Here we showed that KU-0060648 inhibited PI3K-AKT-mTOR activation in HCC cells. Reversely, CA-AKT1 restored AKT-mTOR activation, and inhibited KU-0060648-induced cytotoxicity in HepG2 cells. These results indicate that PI3K-AKT-mTOR in-activation is involved in KU-0060648-exerted actions in HCC cells.
Existing evidences have implied that DNA-PKcs may directly activate AKT-mTOR. For example, Feng and co-authors showed that DNA-PKcs formed a complex with AKT, leading to a 10-fold increase of AKT activity [23]. Dragoi and colleagues demonstrated that activated DNA-PKcs could act as an AKT kinase [42]. Ji et al., displayed that Ultra Violet (UV) radiation induced DNA-PKcs association with mTORC2 component Sin1 to phosphorylate AKT at Ser-473 [10]. In the current study, however, we indicate that PI3K-AKT-mTOR inactivation is unlikely a downstream event of DNA-PKcs inhibition. AKT-mTOR activation was intact in DNA-PKcs-silenced/-overexpressed or DNA-PKcsmutated HepG2 cells. Significantly, KU-0060648 was still anti-proliferative and pro-apoptotic in DNA-PKcs-inhibited/silenced/-mutated HCC cells. Thus, PI3K-AKT-mTOR inhibition could be a direct action by KU-0060648 in HCC cells. This might also explain the superior activity of KU-0060648 in HCC cells, more potently than traditional DNA-PKcs inhibitors (NU-7026, NU-7441).
For many years, HCC has been otherwise a chemoresistant malignancy [3,4]. Molecularly-targeted therapy could be a extremely important option for HCC [3,4]. Our results show that KU-0060648 potently inhibits HCC cells in vitro and in vivo, indicating a possible therapeutic value for HCC.

Culture of established cell lines
Human HCC cell lines, including HepG2, Huh-7 and KYN-2, as well as human HL-7702 hepatocytes were purchased from the Cell Bank of CAS Shanghai (Shanghai, China) at Dec 2014. Cells were maintained in RPMI medium, supplemented with 10% fetal bovine serum (FBS), penicillin/streptomycin, in a humidified incubator. All cell culture reagents were provided by Gibco Life Technologies (Carlsbad, CA). The cell line verification was described in Supplementary Information.

Isolation of human HCC tissues, and culture of primary cells
Surgery-isolated primary human HCC tissues were washed in DMEM. Tumor tissues and surrounding normal liver tissues were separated very carefully under microscopy. A total of eight different primary HCC patients, administered in Wuxi People's Hospital (Wuxi, China), were included in the study (All male, 42-64 years old). These patients received no chemotherapy or radiotherapy prior to surgeries. Fresh tissues were stored in liquid nitrogen. For primary culture of HCC cells, cancer tissues were subjected to collagenase I (Sigma) digestion for 30 min. The resolved single-cell suspensions were then pelleted, washed, and re-suspended in primary cell culture medium (DMEM, 20%-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) [21]. Experiments and protocols requiring human samples were approved by the Internal Review Board (IRB) of all authors' institutions. The writteninformed consent was obtained from each participant. All studies using human samples were conducted according to the principles expressed in the Declaration of Helsinki and according to national and international guidelines.

Chemicals, reagents and antibodies
KU-0060648 was provided by GuideChem (Shanghai, China). The caspase-3 specific inhibitor z-DEVD-fmk and the general caspase inhibitor Z-VADfmk were purchased from Sigma Chemicals (Louis, MO). NU-7026 and NU-7441 were purchased from Calbiochem (San Diego, CA). p-DNA-PKcs (Thr 2609) antibody was purchased from Santa Cruz (Shanghai, China), All other antibodies utilized in this study were obtained from Cell Signaling Tech (Danvers, MA), as described previously [10,22]. The concentrations of agents applied and the treatment durations were chosen based on published literatures and results from pre-experiments.

DNA-PKcs shRNA and stable cell selection
Two non-overlappingDNA-PKcs shRNA lentiviral GV248 plasmids were provided by Dr. Han [18]. These two sets of DNA-PKcs lentiviral-shRNAs were named as DNA-PKcs shRNA Seq-1 and DNA-PKcs shRNA Seq-2. The lentiviral-shRNAs directly added to cultured HepG2 cells (with 60% of confluence, cultured in serumfree medium). After 12 h, virus-containing medium was replaced with fresh complete medium. Stable HepG2 colonies were selected by puromycin (5 μg/mL, Sigma) for 10-12 days. Expression of DNA-PKcs in stable cells was verified by Western blotting.

DNA-PKcs mutation or overexpression
As described [10], the 3-kb HindIII fragment of DNA-PKcs cDNA covering Thr-2609 (Genechem) was utilized as the template for generating the DNA-PKcs T2609A cDNA. The T2609A DNA-PKcs pSV2 neo Flag plasmid [10] or the wild-type (wt-) DNA-PKcs pSV2 neo Flag plasmid (gift from Dr. Li He at Kunming Medical University) [10] was transfected into HepG2 cells with the Lipofectamine 2000 protocol (Invitrogen) [10]. After 48 h, HepG2 cells were re-plated on selection medium containing 100 μg/mL of G418 for 10 days. Stable colonies were isolated, and characterized for expression of DNA-PKcs (Flag-tagged).
Constitutively active-AKT1 (CA-AKT1) expression and stable cell line selection CA-AKT1 vector and the empty vector (Ad-GFP) were reported in our previous study [29]. The plasmid (0.10 μg/mL) was transfected into HepG2 cells with the Lipofectamine 2000 protocol [29]. Stable cells were selected by puromycin (5.0 μg/mL) for 10-12 days. Western blotting was utilized to verify AKT expression/ activation in stable cells.

In vivo anti-tumor efficiency assay
A significant amount of HepG2 cells (5 millions/ mice) were injected subcutaneously into the right flanks of female nude mice (6-8 weeks old). When tumors reached around 100 mm 3 , mice were randomized into three groups with 12 mice per group: vehicle control (saline), 10 mg/ kg of KU-0060648 (intraperitoneal injection or i.p., daily, for 21 days), and 50 mg/kg of KU-0060648 (i.p., daily, for 21 days) [12]. The injection was started when the tumors were established (volumes around 100 mm 3 ). Tumor volumes, recorded every week, were calculated through the established formula: Volume (mm 3 ) = (d 2 × D)/2, in which d and D were the shortest and the longest diameter, respectively. Two weeks after initial KU-0060648 administration, xenografted tumors of two mice per group were isolated, and were subjected to Western blotting and immunohistochemistry (IHC) staining assays. Humane endpoints were applied to minimize suffering. Five weeks after initial KU-0060648 administration, HepG2 xenografts were separated through surgery and weighted. All studies were performed in accordance with the standards of ethical treatment approved by the Institutional Animal Care and Use Committee (IACUC) and Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC).The protocols of the in vivo study were approved by the Animal Care and Use Committee at all authors institutions.
For primary culture of human peripheral blood mononuclear cells (PBMCs) and human skin fibroblasts (HSFs), "Clonogenicity" assay, [H 3 ] Thymidine incorporation assay of cell proliferation, Caspase-3 activity assay, Histone DNA-ELISA assay of cell apoptosis and Western blotting please refer to our previous studies [21,25,[29][30][31]. These methods were described in detail in the Supplementary Information.

Statistical analysis
Data were presented as mean ± standard deviation (SD). Statistics were analyzed by one-way ANOVA followed by a Scheffe' and Tukey Test (SPSS 15.0). Significance was chosen as p < 0.05. IC-50 was calculated by the SPSS software.