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Cathepsin K inhibition-induced mitochondrial ROS enhances sensitivity of cancer cells to anti-cancer drugs through USP27x-mediated Bim protein stabilization

Cathepsin K (Cat K) is expressed in cancer cells, but the effect of Cat K on apoptosis is still elusive. Here, we showed that inhibition of Cat K sensitized the human carcinoma cells to anti-cancer drug through up-regulation of Bim. Inhibition of Cat K increased USP27x expression, and knock down of USP27x markedly blocked Cat K-induced up-regulation of Bim expression. Furthermore, inhibition of Cat K induced proteasome-dependent degradation of regulatory associated protein of mammalian target of rapamycin (Raptor). Down-regulation of Raptor expression increased mitochondrial ROS production, and mitochondria specific superoxide scavengers prevented USP27x-mediated stabilization of Bim by inhibition of Cat K. Moreover, combined treatment with Cat K inhibitor (odanacatib) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) reduced tumor growth and induced cell death in a xenograft model. Our results demonstrate that Cat K inhibition enhances anti-cancer drug sensitivity through USP27x-mediated the up-regulation of Bim via the down-regulation of Raptor.


Introduction
Cathepsin (Cat) K belongs to the papain-like cysteine proteases and is mainly located in the lysosomes [1]. Cat K is originally considered to regulate bone homeostasis in osteoclasts [2]. Cat K is also detected in other cells, such as fibroblasts, chondrocytes, neurons, and glia [3][4][5][6], and Cat K has been shown to be highly expressed in cancer cells [7]. Increased expression of Cats makes cancer cells more sensitive to cathepsin inhibitors. For example, inhibition of Cat S induces reactive oxygen species (ROS)-mediated inhibition of the phosphoinositide 3kinases (PI3K)/Akt signaling and activation of the JNK signaling pathway in human glioblastoma cells [8], and an inhibitor of cysteine cathepsins (Z-Phenylalanine-Glycine-NHO-Bz) induces caspase-and p53-independent cell death in multiple human cancer cells [9]. Similarly, a dual inhibitor of Cat B and L (Fmoc-Tyr-Ala-CHN 2 ) increases cell death in neuroblastoma [10] and an inhibitor of Cats B, L, S and V (VBY-825) decreases tumor burden and tumor number in a RIP1-Tag2 mouse model of pancreatic neuroendocrine cancer [11]. In other cancers, inhibition of Cats alone has no effect on apoptosis but increases cell sensitivity to anti-cancer drugs. Inhibition of Cat S and Cat G enhances TRAIL-mediated apoptosis through the modulation of the expression of anti-apoptotic proteins [12,13], and inhibition of Cat L increases the rate of irradiation-mediated apoptosis [14,15]. In addition, inhibition of Cats enhances anti-cancer effects, including induction of apoptosis and inhibition of invasion and cell growth [16][17][18][19][20][21]. Although Cat K modulates invasion and metastasis through degradation of matrix proteins in melanoma [22], it is still unclear whether Cat K plays additional roles in cancer cells. mTOR signaling is highly activated in cancer cells. mTOR forms two distinct multiprotein complexes, mTORC1 and mTORC2 [23,24]. mTORC1 is composed of mTOR, regulatory associated protein of mammalian target of rapamycin (Raptor), G protein beta protein subunit-like (GβL), DEP domain-containing mTOR-interacting protein (DEPTOR), and proline-rich Akt substrate of 40 kDa (PRAS40), and it regulates phosphorylation of p70 ribosomal protein S6 kinase 1 (S6K) and eukaryotic initiation factor 4E binding protein 1 (4EBP1). mTORC2 includes mTOR, GβL, rapamycin-insensitive companion of mammalian target of rapamycin (Rictor), Sin1, proline-rich protein 5 (PRR5)/PRR5like (L), and DEPTOR and modulates phosphorylation of Akt and protein kinase C (PKC) [23,24]. Activation of mTORC1/2 regulates cell growth, migration, invasion, and metastasis in cancer cells [25]. The importance of the mTOR signaling in cancer cells is emphasized by the fact that its activation is critical for drug resistance. Inhibitors of RAF or MEK are used for the treatment of BRAF-mutant melanoma; however, their successful action is dependent on the inhibition of TORC1/2 activity. Sustained TORC1 activity induces drug resistance [26], and inhibition of mTORC1/2 sensitizes BRAF-mutant melanoma to MEK inhibitor-induced apoptosis [27]. Up-regulation of Raptor also contributes to resistance to the treatment with a PI3K-mTOR inhibitor in renal cancer cells [28]. In addition, inhibition of the PI3K/Akt/mTOR signaling overcomes resistance to trastuzumab-treated HER2-targeted therapy in breast cancer cells [29] and to everolimus in pancreatic neuroendocrine tumor cells [30]. Thus, modulation of the mTOR signaling can be a targeted treatment with a dual effect in cancer cells: it can negatively interfere with the onset of cancer and suppress anticancer resistance.
In view of this evidence, in this study, we investigated the effect of Cat K inhibition on cell death, and the related molecular mechanisms were evaluated in human renal carcinoma Caki cells. The indicated cancer cell lines were transfected with control siRNA (siCont) or Cat K siRNA (siCat K) and treated with 50 ng/mL TRAIL for 24 h. Apoptosis and protein expression were measured by flow cytometry and western blotting. The values in the graphs (A-E) represent the mean ± SD of three independent experiments. *p < 0.01 compared to the control.

Knockdown of gene by siRNA
The siRNA transfected into cells using Lipofectamine RNAiMAX (Thermo Fisher Scientific, Waltham, MA, USA).

Analysis of cell cycle and apoptosis using flow cytometry
Cells were fixed with 100% ethanol for 2 h, incubated in 1.12% sodium citrate buffer containing 50 μg/mL RNase for 30 min at 37°C, added to 50 μg/mL propidium iodide, and measured using the BD Accuri™ C6 Cytometer (BD Biosciences, San Jose, CA, USA).

Western blotting
Western blotting was performed according to methods described in our previous study [32]. In brief, the lysates were collected, boiled with 5X sample buffer, and separated by SDS-PAGE. Proteins on membrane were probed with specific antibodies, and the antibodies were detected by enhanced chemiluminescence (ECL) solution (EMD Millipore, Darmstadt, Germany).

Detection of apoptosis by DNA fragmentation and DEVDase activity assay
Caki cells were treated with ODN alone, TRAIL alone or combinations of ODN plus TRAIL for 24 h. To check the change of cellular nuclei, cells were fixed with 1% paraformaldehyde and added to 4′,6′diamidino-2-phenylindole solution for 5 min (Roche, Basel, Switzerland). The change of cell morphology and condensation of the nucleus were examined by fluorescence microscope (Carl Zeiss, Jena, Germany). DNA fragmentation assay was used for cell death detection ELISA plus kit (Roche, Basel, Switzerland). For DEVDase activity assay, cells were harvested and incubated with reaction buffer containing acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA) substrate. Cell morphology (B) and nuclear condensation (C) were assesses using a microscope. Quantification of DNA fragments was determined using a DNA fragmentation assay kit (D). Detection of caspase activity was measured using a DEVDase colorimetric assay kit (E). (F) Caki cells were treated with 2 μM ODN and 50 ng/mL TRAIL in the presence or absence of a pan-caspase inhibitor, z-VAD-fmk (20 μM), for 24 h. Apoptosis and protein expression were measured by flow cytometry, and western blotting, respectively. The values in the graphs (A and D-F) represent the mean ± SD of three independent experiments. *p < 0.01 compared to the control. #p < 0.01 compared to the combinations of ODN and TRAIL.

Ubiquitination assay
This assay was described using the tagged-ubiquitin plasmid and pretreatment of MG132 as previously report [35]. Briefly, cells were harvested, washed with PBS containing 10 mM N-ethylmaleimide (NEM) (EMD Millipore, Darmstadt, Germany), resuspended in 90 μL PBS/NEM containing 1% SDS, and boiled for 10 min at 95°C. Lysates were added to lysis buffer involving 1 mM PMSF and 5 mM NEM, dissolved using l mL syringe for 3-4 times and centrifuged at 13,000×g for 10 min at 4°C. The supernatants were incubated with primary antibody of target protein for overnight and reacted by adding protein G agarose bead for 2 h. After centrifuging, the supernatants were removed, washed with lysis buffer containing 1 mM PMSF and 5 mM NEM at 2 times and boiled using 2X sample buffer for 10 min. Ubiquitinated Raptor and Bim were detected using HRP-conjugated anti-Ub.

Construct of stable cell lines by transfection
To construct the stable cell lines, pDsRed2-Mito vector plasmids transiently transfected into Caki cells using Lipofactor-pMAX (Aptabio, Yongin, Korea). After 2 days, cells were replaced with fresh media and selected by the G418 (700 μg/mL) (Invitrogen, Carlsbad, CA, USA). After 3 weeks, red fluorescence of labeling of mitochondria was detected by fluorescence microscope.

Analysis of mitochondrial lengths
Caki/pDsRed2-Mito cells were treated with 2 μM ODN for 6 h. Fluorescence images of mitochondrial morphology was analyzed by Confocal Laser Microscope (Carl Zeiss, Jena, Germany). Mitochondrial . (E) Caki cells were transfected with siCont or Bim siRNA (siBim) and treated with/without 2 μM ODN plus 50 ng/mL TRAIL for 24 h. Apoptosis and protein expression were measured by flow cytometry and western blotting, respectively. The values in the graphs (B and E) represent the mean ± SD of three independent experiments. *p < 0.01 compared to the control. †p < 0.01 compared to the combinations of ODN and TRAIL in siCont. lengths were measured using LSM 5 Image Browser. In 3 independent experiments, average lengths of at least five mitochondria were analyzed from randomly selected areas for each data. Showing images were obtained from differences between six individual images.

Detection of mitochondrial damage
For mitochondrial damage, Caki cells were stained to MitoTracker Deep Red and MitoTracker Green dye (Molecular Probes Inc., Eugene, OR, USA) for 15 min after treatment of ODN for 6 h. Cells were trypsinized and resuspended 300 μL of PBS. Mitochondrial damage was measured using the FACSCanto™ flow cytometer (BD Biosciences, San Jose, CA, USA).

ATP production assay
Detection of ATP levels were analyzed using ATP determination kit (A22066, Thermo Fisher Scientific, Waltham, MA, USA). Caki cells were treated with ODN for 6 h. After, cells were harvested, washed with cold PBS and lysed in provided lysis buffers in the kits according to the manufacturer's instructions. After centrifuging, the supernatants were mixed with standard reaction buffer in 96 well microplates and incubated for 15 min at room temperature. ATP production was measured by luminescence using Infinite® 200 PRO microplate reader (Tecan, Männedorf, Switzerland).

Measurement of mitochondrial ROS
To measure mitochondrial ROS production, cells were determined using the MitoSOX Red mitochondrial superoxide indicator (Thermo (caption on next page) Fisher scientific, Waltham, MA, USA). Before the harvest of lysate, cells were stained with the MitoSOX Red dye for 10 min. And then cells were trypsinized and resuspended in PBS, and mitochondrial ROS production was measured by red fluorescence using the BD Accuri™ C6 Cytometer (BD Biosciences, San Jose, CA, USA).

Animal
Male BALB/c-nude mice, aged 5 weeks, were purchased from the Central Lab Animal Inc. (Seoul, Korea). All the mice were allowed 1 week to acclimatize to the surroundings before the experiments, and were kept at 25 ± 2°C, with a relative humidity of 55 ± 5% and a 12 h light-dark cycle. The study protocol was approved by the IRB Keimyung University Ethics Committee.

In vivo xenograft model and detection of TUNEL assay
Development of xenograft models were previously described in our previous study [36]. Experiment grope were divided by vehicle alone, 5 mg/kg ODN (20% DMSO + PBS) alone, 3 mg/kg GST-TRAIL alone, and in combinations of ODN and GST-TRAIL for 24 days. For apoptosis in vivo, TUNEL assay was performed according to methods described in our previous study [36].

Statistical analysis
We repeated experiments in our studies at least three times, and all data are represented as the means. Statistical analysis was performed by a one-way ANOVA and post hoc comparisons (Student-Newman-Keuls) using the SPSS (Statistical Package for the Social Sciences, version 22.0) (SPSS Inc.; Chicago, IL). We decide the sample size on the basis of the minimum effects we wish to measure. The p-values < 0.05 were considered significant.

Knockout and knockdown of Cat K sensitize the cancer cells to anticancer drugs
Since it has been known that Cats are highly expressed in cancer cells compared with normal cells [7], we investigated the effect of Cat K inhibition on cancer cell death. To evaluate the effects of Cat K genomic deletion in human renal carcinoma Caki cells, we used the CRISPR-Cas9 genome editing system [31]. The effect of Cat K knockout (KO) on apoptosis was minimal until five days (Caki/Cat K KO; Fig. 1A). Therefore, we investigated whether inhibition of Cat K sensitizes the cells to anti-cancer drugs. The Cat K inhibitor odanacatib (ODN) alone had no effect on apoptosis, but the combined treatment with ODN and a sub-lethal dose of anti-cancer drugs markedly induced apoptosis (Fig. 1B). We also confirmed the chemosensitizing effect of Cat K inhibition using Caki/Cat K KO cells. Sub-lethal doses of TRAIL, a cancer cell-specific inducer of apoptosis, had no effect on the apoptosis in Caki cells, but apoptosis and cleaved PARP (being PARP a substrate of caspase-3) were markedly increased in TRAIL-treated Caki/Cat K KO cells (Fig. 1C). In addition, sub-lethal doses of other cancer drugs (doxorubicin, etoposide, 5FU, cisplatin, and oxaliplatin) also induced apoptosis in Cat K KO cells (Fig. 1D). Using Cat K RNA interference in other cancer cells (human renal carcinoma ACHN, human head and neck carcinoma AMC-HN4, and human cervical carcinoma Hela cells), we found that the sensitizing effect of Cat K inhibition to TRAIL-mediated apoptosis was a common response (Fig. 1E). Therefore, inhibition of Cat K sensitizes cancer cells to anti-cancer drugs.

Cat K inhibitors enhance TRAIL-induced apoptosis via caspase-3 activation
Next, we tested whether Cat K inhibitors could mimic anti-cancer effect of cathepsin K knockdown (KD) or knockout (KO). Two Cat K inhibitors, ODN and balicatib, dose-dependently induced apoptosis and PARP cleavage in TRAIL-treated Caki cells ( Fig. 2A), and combined treatment with ODN and TRAIL induced apoptosis-related morphological changes, nuclear chromatin damage, and DNA fragmentation (Fig. 2B-D). Furthermore, combined treatment markedly activated caspase-3 (Fig. 2E). The pan-caspase inhibitor (z-VAD) completely blocked apoptosis (Fig. 2F). Cat K inhibitors also sensitized the cells to TRAIL.
3.3. Up-regulation of Bim expression plays a critical role in the enhancement of the cytotoxicity of anti-cancer drugs Next, we wondered which molecular mechanisms were associated with the increased sensitivity to anti-cancer drugs upon inhibition of Cat K. When we screened for apoptosis-related proteins whose levels changed upon ODN treatment, Bim and Bax expression was increased, whereas other proteins were not altered ( Fig. 3A and B). Unlike ODN, balicatib induced down-regulation of c-FLIP and Mcl-1 expression, and up-regulation of Bim and Bax expression ( Fig. 3A and B). Since KD and KO of Cat K did not reduce expression of c-FLIP and Mcl-1, but increased Bim and Bax expression ( Fig. 3C and D). Down-regulation of c-FLIP and Mcl-1 by balicatib might be independent of inhibition of Cat K. To validate the role of Bim up-regulation by ODN, cells were transfected with Bim siRNA. Knockdown of Bim decreased ODN plus TRAIL-induced apoptosis and PARP cleavage (Fig. 3E), and balicatib plus TRAILinduced apoptosis is also inhibited by knockdown of Bim ( Supplementary Fig. S1). For further studies, we focused on up-regulation of Bim using ODN. These results suggested that up-regulation of Bim expression plays a critical role in the enhancement of the cytotoxicity of TRAIL by Cat K inhibition.

USP27x stabilizes Bim
Within 6 h from the treatment, ODN increased Bim expression at the protein, but not at the mRNA level (Fig. 4A). Therefore, we investigated whether ODN modulates Bim expression at the post-translational level. Caki cells were treated with or without ODN in the presence of cycloheximide (CHX; inhibitor of de novo protein synthesis) for various periods. ODN significantly enhanced Bim stability compared with CHX alone (Fig. 4B). Next, to identify the molecular mechanism, which modulates protein stability of Bim, we investigated the expression of E3 ligases (TRIM2, β-TrCP, and Cdc20) and deubiquitinases (USP9x and USP27x) of Bim [37][38][39][40][41]. ODN did not effect on expression levels of TRIM2, β-TrCP, Cdc20 and USP9x ( Fig. 4C and D). However, ODN only increased USP27x expression within 3 h (Fig. 4D), and KD or KO of Cat K also induced USP27x expression ( Fig. 4E and F). ODN had no effect on USP27x mRNA expression ( Supplementary Fig. S2). Furthermore, we found that ODN-induced up-regulation of USP27x expression is related with Bim stabilization (Fig. 4G). In addition, ODN dramatically inhibited ubiquitination of Bim, but KD of USP27x blocked ODN-induced deubiquitination of Bim (Fig. 4H). These data indicated that inhibition of Cat K increases Bim stabilization via up-regulation of USP27x expression.

Down-regulation of raptor is a critical role for Bim stabilization in ODN -treated cells
Next, we elucidated the molecular mechanisms underlying the stabilization of Bim upon Cat K inhibition. We investigated modulation of various kinases phosphorylation to find signaling pathways controlled by ODN, and we found ODN inhibited mTORC1 signaling, which is known to inhibit the expression of Bim [42]. As shown in Fig. 5A, ODN significantly reduced the phosphorylation of mTOR at S2448 (Fig. 5A), which is related with mTORC1 signaling [43]. In addition, the expression of Raptor and PRAS40, and the phosphorylation of S6K and 4EBP1 (substrates of mTORC1) were also markedly reduced by ODN (Fig. 5A). In contrast, the expression and phosphorylation of mTORC2related proteins, and the expression of mTORC1/2 common proteins did not significantly change (Fig. 5A). Therefore, mTORC1 signaling pathway may regulate Bim expression through inhibition of Cat K. Since Raptor is a critical for the binding of mTORC1 substrates [44] and dissociation of Raptor from mTORC1 inhibits mTOR signaling [45], we first checked the role of Raptor. Overexpression of Raptor reversed ODN-mediated dephosphorylation of mTOR (S2448), S6K, 4EBP1 (Fig. 5B), and inhibited the stabilization of Bim and resulted in inhibition of ODN plus TRAIL-induced apoptosis and PARP cleavage (Fig. 5C). PRAS40, which has an inhibitory effect of mTORC1 kinase activity [46], was down-regulated by ODN (Fig. 5A). However, PRAS40 overexpression did not inhibit ODN plus TRAIL-induced apoptosis and stabilization of Bim (Fig. 5D).
To investigate further the molecular mechanisms underlying the down-regulation of Raptor upon Cat K inhibition, we analyzed the transcriptional regulation of Raptor. The expression levels of Raptor mRNA did not change in ODN-treated cells (Fig. 5E). We then investigated whether ODN regulated Raptor at the post-translational level. Treatment with a proteasome inhibitor (MG132) rescued ODNinduced down-regulation of Raptor expression (Fig. 5F), and the combined treatment with CHX and ODN markedly reduced Raptor stability, compared with the treatment with CHX alone (Fig. 5G). Since the levels of Raptor are regulated through ubiquitination [47,48], we tested the effect of ODN on the ubiquitination of Raptor. ODN markedly increased polyubiquitination of Raptor (Fig. 5H). Therefore, our data suggested that ODN decreases the stability of Raptor at the protein level. We also detected up-regulation of Bim and USP27x expression and down-regulation of Raptor by pharmacological Cat K inhibitor (ODN) and Cat K siRNA in other cancer cell lines ( Fig. 5I and J), Therefore, these data suggested that Cat K inhibition induces stabilization of Bim through down-regulation of Raptor.

Down-regulation of raptor inhibits mitochondrial fission
We investigated how down-regulation of Raptor by ODN sensitizes the cells to anti-cancer drugs. Since mTORC1 signaling is a key regulator of mitochondrial dynamics [49], we first focused on this process. ODN increased the number of elongated mitochondria (> 5 μm), whereas the number of fragmented (< 2 μm) and intermediate (2-5 μm) mitochondria decreased (Fig. 6A). ODN inhibited phosphorylation of Drp1 at S616, related with pro-fission activity (Fig. 6B) [50][51][52]. Furthermore, using two dyes that distinguish respiring mitochondria (MitoTtracker Deep Red) and total mitochondria (Mito-Tracker Green) [53], we found that damaged mitochondria increased (Fig. 6C). ODN-induced mitochondrial damage decreased ATP production and increased mitochondrial ROS production ( Fig. 6D and E). We then investigated whether mitochondrial ROS are involved in ODN plus TRAIL-induced apoptosis. A blocker of mitochondrial ROS (Mito-TEMPO) significantly inhibited apoptosis and PARP cleavage (Fig. 6F). Up-regulation of Bim expression was also reversed by Mito-TEMPO treatment, while Mito-TEMPO failed to reverse the expression of Raptor in ODN-treated cells (Fig. 6G), indicating that Raptor is an upstream signaling molecule for mitochondrial ROS. To prove this hypothesis, we overexpressed Raptor in Caki cells. Overexpression of Raptor markedly inhibited mitochondrial ROS production, and reversed dephosphorylation of Drp1 at S616 in ODN-treated cells (Fig. 6H and I). Furthermore, overexpression of Raptor also reduced the number of elongated mitochondria (> 5 μm) in ODN-treated cells (Fig. 6J). Next, we investigated whether mitochondrial ROS is related with mitochondrial fusion. Blockers of mitochondrial ROS (Mito-TEMPO and MnTMPyP) inhibited ODN-induced dephosphorylation of Drp1 (Fig. 6K). In addition, overexpression of Raptor and blockers of mitochondrial ROS blocked ODN-induced USP27x expression (Fig. 6L and  M). These data suggested that Raptor downregulation-mediated mitochondrial ROS production is critical for the enhancement of ODNinduced anti-cancer drugs sensitivity via USP27x-mediated Bim stabilization. Fig. 6. Downregulation of Raptor expression induces mitochondrial ROS production. (A) Representative confocal images obtained on cells stably transfected with a pDsRed2-mito (red) plasmid labeling mitochondria after treatment with 2 μM ODN for 6 h. The nuclei were stained with DAPI (blue), and the length of the mitochondria was measured using LSM 5 Image Browser. (B-E) Caki cells were treated with 2 μM ODN for the indicated time (B and E) or 6 h (C and D) and analyzed for indicated proteins by western blotting (B). Flow cytometry was used to detect fluorescence intensity for mitochondrial damage (C) and ATP production was measured using an ATP determination kit (D). Flow cytometry was used to detect fluorescence intensity for mitochondrial ROS (E). (F and G) Caki cells were pretreated with different concentrations of Mito-TEMPO and treated with 2 μM ODN and 50 ng/mL TRAIL (F) or 2 μM ODN (G) for 24 h, and apoptosis and protein expression were measured by flow cytometry and western blotting, respectively. (H and I) Caki cells were transfected with pRK5 (Caki/Vec) or pRK5-Myc-Raptor (Caki/Raptor) and treated with 2 μM ODN for 6 h (H) or 24 h (I). Mitochondrial ROS production was analyzed by MitoSOX Red reagent staining and flow cytometry. (J) Representative confocal images obtained on Caki/pDsRed2-mito cells transfected with pRK5 (Vec) or pRK5-Myc-Raptor (Raptor) after treatment with 2 μM ODN for 6 h. The nuclei were stained with DAPI, and the length of the mitochondria was measured using LSM 5 Image Browser.

Combined treatment with ODN and TRAIL reduces tumor growth in vivo
Finally, we examined the effect of the combined treatment with ODN and TRAIL in xenograft model. ODN plus TRAIL markedly suppressed tumor growth, compared with the other conditions ( Fig. 7A and  B), and increased TUNEL-positive staining (Fig. 7C). Furthermore, ODN also induced up-regulation of Bim and down-regulation of Raptor expression (Fig. 7D). In addition, combined treatment with ODN and TRAIL had no effect on cell death in normal mouse kidney cells (TCMK-1) and normal human mesangial cells (MC) (Fig. 7E). ODN did not induce up-regulation of Bim expression and down-regulation of Raptor expression in mesangial cells, while Raptor and Bim proteins were undetected in TCMK-1 cells (Fig. 7F). These results suggested that inhibition of Cat K sensitizes the cells to anti-cancer drug-induced apoptosis, and up-regulation of Bim expression is involved in the enhancement of the cytotoxicity of anti-cancer drugs.

Discussion
In the present study, we demonstrated that inhibition of Cat K enhanced the chemosensitivity of cancer cells. Down-regulation of Raptor by ODN increased mitochondrial ROS levels, and inhibited mitochondria fission via regulation of Drp1 phosphorylation at S616. Furthermore, ODN-mediated mitochondrial ROS production induced USP27x expression. Up-regulation of USP27x by ODN is associated with the up-regulation of Bim expression, which is critical for ODN-induced chemosensitivity of cancer cells (Fig. 8). We also revealed that combined treatment with ODN and TRAIL reduced tumor size and increased apoptosis in a xenograft model (Fig. 7A-C). There are many evidences about the links between levels of ROS and mitochondrial dynamics. In general, ROS induces mitochondrial fission via multiple pathways [54]. However, ODN-induced ROS promoted mitochondria fusion via dephosphorylation of Drp1 at S616 (Fig. 6K). Morita et al. reported that mitochondrial fission process protein 1 (MTFP1) plays a critical role in Drp1 phosphorylation at S616 during mTORC1 inhibition [49]. Inhibition of mTOR reduces MTFP1 expression, resulting in inhibition of Drp1 phosphorylation at S616. However, they did not find how MTFP1 regulates DRP1 phosphorylation at S616. We thought that ROS are one of regulators, which induces dephosphorylation of Drp1 at S616 during mTORC1 inhibition, and there is a possibility that MTFP1 will be involved in ODN-induced dephosphorylation of Drp1 at S616. However, we need further study to identify how ROS modulates mitochondrial dynamic via modulation of Drp1 phosphorylation at S616.
Inhibition of Cat K increased Bim expression in multiple cancer cells lines (Fig. 3C, D, 5I, and 5J). Up-regulation of Bim expression by ODN was not detected at the transcriptional level (Fig. 4A), and stability of Bim protein was increased in ODN-treated cells (Fig. 4B). Stability of Bim protein is modulated via ubiquitin-proteasome pathway. For examples, Cdc20 induces Bim ubiquitination, resulting in degradation of Bim [39]. Tripartite motif protein 2 (TRIM2) and β-TrCP also induced ubiquitination of Bim in an ERK MAPK-and Rsk1/2-dependent manner, respectively [37,38]. We examined whether ODN modulates E3 ligases of Bim in Caki cells, but ODN did not change the expression of E3 ligases, such as TRIM2, β-TrCP, and Cdc20 (Fig. 4C). Therefore, we focused on DUBs of Bim. As shown in Fig. 4D, ODN markedly increased USP27x expression, but not USP9x. Furthermore, KD of USP27x blocked ODN-induced Bim expression and deubiquitination of Bim ( Fig. 4G and H). Weber et al. reported that USP27x stabilizes Bim and induces apoptosis [41]. Phosphorylation of Bim by Raf-ERK pathway increases ubiquitination in a β-TrCP dependent manner, resulting in degradation of Bim. USP27x had no affect the phosphorylation of Bim, but increases stability of Bim by inhibition of ubiquitination [41]. In addition, USP27x is DUB of Snail1 [55]. Snail1 is a critical transcription factor to modulate epithelial-to-mesenchymal transition (EMT)-related genes and is related with resistance to chemotherapy. USP27x is increased TGF-β, which is induces EMT, and stabilized Snail1 [55]. Furthermore, USP27x interacts with cyclin E, resulting in stabilization [56]. Treatment with 5-fluorouracil (5-FU) decreases USP27x expression, and then induces degradation of cyclin E [56]. In our study, ODN also increased USP27x expression (Fig. 4D). The regulation of USP27x expression might be important on modulation of stability of substrates. Therefore, modulatory mechanism of USP27x expression also need further detailed investigation.
In case of Raptor, inhibition of Cat K also markedly induced downregulation of Raptor expression (Fig. 5A). As shown in Fig. 5E-G, downregulation of Raptor was not detected at the transcriptional level, and inhibition of Cat K decreased the stability of Raptor in a proteasomedependent manner. There are few reports on the regulation of Raptor expression at the post-translational level. Bridges et al. reported that USP9x directly binds to Raptor and inhibits its proteasomal degradation, resulting in activation of the mTORC1 signaling in neural progenitor cells [48]. However, in our system, ODN had no effect on USP9x expression and KD of USP9x did not inhibit Raptor expression ( Fig. 4D and Negative data; Data not shown). In addition, UCHL1 inhibits the ubiquitination of Raptor [47]. However, KD of UCHL1 inhibits ubiquitination of Raptor by DDB1-CUL4, resulting in instability of mTORC1. Deubiquitination of Raptor by UCHL1 did not change stability of Raptor. Antagonizing DDB1-CUL4-mediated ubiquitination of Raptor by UCHL1 decreases mTORC1 signaling, but activates mTORC2 signaling. Therefore, we could rule out the UCHL1 as a DUB of Raptor. We have identified several possibilities to determine the regulatory mechanisms of the protein stability of Bim and Raptor by inhibition of Cat K, but further research needs to validate. For renal cell carcinoma patients, inhibitors of multi-targeted tyrosine kinase, VEGF receptor, and mTOR are treated to improve the survival [57]. However, resistance to these drugs alleviates the anti-cancer effects. Interestingly, Earwaker et al. studied how renal carcinoma cells acquire resistance to PI3K/mTOR inhibitor. They suggested that drug resistance is correlated with Raptor expression [28]. In our study, inhibition of Cat K sensitized cancer cells to multiple anti-cancer drugs (Fig. 1B), but overexpression of Raptor completely blocked ODN-induced sensitization cancer cells to TRAIL (Fig. 5C). Previous studies reported that mTOR signaling pathway is associated with mitochondria functions. For examples, complex formation of mTOR and raptor is correlated with mitochondrial oxygen consumption and oxidative capacity [58], and mTOR controls mitochondrial oxygen consumption through the complex formation with a yin-yang 1 (YY1), peroxisome-proliferator activated receptor coactivator (PGC)-1α, and mTORC1 [59]. Furthermore, MTFP1 has been known to regulate mTORC1-mediated modulation of mitochondria dynamic [49]. In our study, inhibition of Cat K also induced mitochondrial dysfunction and ROS production (Fig. 6C-E), and overexpression of Raptor prevented mitochondrial damage by inhibition of Cat K (Fig. 6H and I). Inhibition of mTORC1 by inhibitors is transient [60], thus degradation of Raptor by ODN might have more effective inhibitory function on mTORC1, because raptor is critical for activation of mTORC1 signaling as a scaffold protein. Therefore, down-regulation of raptor by inhibition of Cat K could be effective strategy for anticancer drug resistant cancer cells therapy.
Taken together, we showed that Cat K inhibition sensitizes the cells to anti-cancer drugs through the up-regulation of USP27x-mediated Bim expression by down-regulation of Raptor. These observations suggest that inhibition of Cat K sensitizes cancer cells to anti-cancer drugs.

Declaration of competing interest
The authors declare that they have no conflict of interest.