USP41 Enhances Epithelial-Mesenchymal Transition of Breast Cancer Cells through Snail Stabilization.

Ubiquitination, one of many post-translational modifications, causes proteasome-mediated protein degradation by attaching ubiquitin to target proteins. Multiple deubiquitinases inhibit the ubiquitination pathway by removing the ubiquitin chain from protein, thus contributing to the stabilization of substrates. USP41 contributes to invasion, apoptosis and drug resistance in breast and lung cancer cells. However, the detailed mechanism and role of USP41 in breast cancer have not been elucidated. USP41 was overexpressed and showed poor prognosis according to the aggressive phenotype of breast cancer cells. Knockdown of USP41 inhibited migration and growth of breast cancer cells, whereas overexpression of USP41 increased cell growth and migration. In addition, depletion of USP41 downregulated Snail protein expression, an epithelial-mesenchymal transition marker, but not mRNA expression. Furthermore, USP41 interacted with and inhibited ubiquitination of Snail, resulting in the increase in Snail stabilization. Therefore, these data demonstrated that USP41 increases migration of breast cancer cells through Snail stabilization.

Tubeimoside-1 Enhances TRAIL-Induced Apoptotic Cell Death through STAMBPL1-Mediated c-FLIP Downregulation.

Tubeimoside-1 (TBMS-1), a traditional Chinese medicinal herb, is commonly used as an anti-cancer agent. In this study, we aimed to investigate its effect on the sensitization of cancer cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Our results revealed that even though monotherapy using TBMS-1 or TRAIL at sublethal concentrations did not affect cancer cell death, combination therapy using TBMS-1 and TRAIL increased apoptotic cell death. Mechanistically, TBMS-1 destabilized c-FLIP expression by downregulating STAMBPL1, a deubiquitinase (DUB). Specifically, when STAMBPL1 and c-FLIP bound together, STAMBPL1 deubiquitylated c-FLIP. Moreover, STAMBPL1 knockdown markedly increased sensitivity to TRAIL by destabilizing c-FLIP. These findings were further confirmed in vivo using a xenograft model based on the observation that combined treatment with TBMS-1 and TRAIL decreased tumor volume and downregulated STAMBPL1 and c-FLIP expression levels. Overall, our study revealed that STAMBPL1 is essential for c-FLIP stabilization, and that STAMBPL1 depletion enhances TRAIL-mediated apoptosis via c-FLIP downregulation.

Inhibition of USP2 Enhances TRAIL-Mediated Cancer Cell Death through Downregulation of Survivin.

Ubiquitin-specific protease 2 (USP2) is a deubiquitinase belonging to the USPs subfamily. USP2 has been known to display various biological effects including tumorigenesis and inflammation. Therefore, we aimed to examine the sensitization effect of USP2 in TRAIL-mediated apoptosis. The pharmacological inhibitor (ML364) and siRNA targeting USP2 enhanced TNF-related apoptosis-inducing ligand (TRAIL)-induced cancer cell death, but not normal cells. Mechanistically, USP2 interacted with survivin, and ML364 degraded survivin protein expression by increasing the ubiquitination of survivin. Overexpression of survivin or USP2 significantly prevented apoptosis through cotreatment with ML364 and TRAIL, whereas a knockdown of USP2 increased sensitivity to TRAIL. Taken together, our data suggested that ML364 ubiquitylates and degrades survivin, thereby increasing the reactivity to TRAIL-mediated apoptosis in cancer cells.

Melatonin induces apoptotic cell death through Bim stabilization by Sp1-mediated OTUD1 upregulation.

Melatonin, secreted by the pineal gland, regulates the circadian rhythms and also plays an oncostatic role in cancer cells. Previously, we showed that melatonin induces the expression of Bim, a pro-apoptotic Bcl-2 protein, at both the transcriptional and post-translational levels. In the present study, we investigated the molecular mechanisms underlying the melatonin-mediated Bim upregulation through post-translational regulation. We found that ovarian tumor domain-containing protein 1 (OTUD1), a deubiquitinase belonging to the OTU protein family, is upregulated by melatonin at the mRNA and protein levels. OTUD1 knockdown inhibited melatonin-induced Bim upregulation and apoptosis in cancer cells. OTUD1 directly interacted with Bim and inhibited its ubiquitination. Melatonin-induced OTUD1 upregulation caused deubiquitination at the lysine 3 residue of Bim, resulting in its stabilization. In addition, melatonin-induced activation of Sp1 was found to be involved in OTUD1 upregulation at the transcriptional level, and pharmacological inhibition and genetic ablation of Sp1 (siRNA) interrupted melatonin-induced OTUD1-mediated Bim upregulation. Furthermore, melatonin reduced tumor growth and induced upregulation of OTUD1 and Bim in a mouse xenograft model. Notably, Bim expression levels correlated with OTUD1 levels in patients with renal clear cell carcinoma. Thus, our results demonstrated that melatonin induces apoptosis by stabilizing Bim via Sp1-mediated OTUD1 upregulation.

Inhibition of BMI-1 Induces Apoptosis through Downregulation of DUB3-Mediated Mcl-1 Stabilization.

BMI-1, a polycomb ring finger oncogene, is highly expressed in multiple cancer cells and is involved in cancer cell proliferation, invasion, and apoptosis. BMI-1 represents a cancer stemness marker that is associated with the regulation of stem cell self-renewal. In this study, pharmacological inhibition (PTC596) or knockdown (siRNA) of BMI-1 reduced cancer stem-like cells and enhanced cancer cell death. Mechanistically, the inhibition of BMI-1 induced the downregulation of Mcl-1 protein, but not Mcl-1 mRNA. PTC596 downregulated Mcl-1 protein expression at the post-translational level through the proteasome-ubiquitin system. PTC596 and BMI-1 siRNA induced downregulation of DUB3 deubiquitinase, which was strongly linked to Mcl-1 destabilization. Furthermore, overexpression of Mcl-1 or DUB3 inhibited apoptosis by PTC596. Taken together, our findings reveal that the inhibition of BMI-1 induces Mcl-1 destabilization through downregulation of DUB3, resulting in the induction of cancer cell death.

Lucanthone, Autophagy Inhibitor, Enhances the Apoptotic Effects of TRAIL through miR-216a-5p-Mediated DR5 Upregulation and DUB3-Mediated Mcl-1 Downregulation.

A lucanthone, one of the family of thioxanthenones, has been reported for its inhibitory effects of apurinic endonuclease-1 and autophagy. In this study, we investigated whether lucanthone could enhance tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in various cancer cells. Combined treatment with lucanthone and TRAIL significantly induced apoptosis in human renal carcinoma (Caki and ACHN), prostate carcinoma (PC3), and lung carcinoma (A549) cells. However, combined treatment did not induce apoptosis in normal mouse kidney cells (TCMK-1) and normal human skin fibroblast (HSF). Lucanthone downregulated protein expression of deubiquitinase DUB3, and a decreased expression level of DUB3 markedly led to enhance TRAIL-induced apoptosis. Ectopic expression of DUB3 inhibited combined treatment with lucanthone and TRAIL-induced apoptosis. Moreover, lucanthone increased expression level of DR5 mRNA via downregulation of miR-216a-5p. Transfection of miR-216a-5p mimics suppressed the lucanthone-induced DR5 upregulation. Taken together, these results provide the first evidence that lucanthone enhances TRAIL-induced apoptosis through DR5 upregulation by downregulation of miR-216a-5p and DUB3-dependent Mcl-1 downregulation in human renal carcinoma cells.

Axl Inhibitor R428 Enhances TRAIL-Mediated Apoptosis Through Downregulation of c-FLIP and Survivin Expression in Renal Carcinoma.

R428, a selective small molecule Axl inhibitor, is known to have anti-cancer effects, such as inhibition of invasion and proliferation and induction of cell death in cancer cells. The Axl receptor tyrosine kinase is highly expressed in cancer cells and the level of Axl expression is associated with survival, metastasis, and drug resistance of many cancer cells. However, the effect of Axl inhibition on overcoming anti-cancer drugs resistance is unclear. Therefore, we investigated the capability of Axl inhibition as a therapeutic agent for the induction of TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) sensitivity. In this study, R428 markedly sensitized cancer cells to TRAIL-induced apoptotic cell death, but not in normal human skin fibroblast (HSF) and human umbilical vein cells (EA.hy926). Moreover, knockdown of Axl by siRNA also increased TRAIL-induced apoptosis. R428 decreased c-FLIP proteins levels via induction of miR-708 expression and survivin protein levels at the post-translational level, and we found that knockdown of Axl also decreased both c-FLIP and survivin protein expression. Overexpression of c-FLIP and survivin markedly inhibited R428 plus TRAIL-induced apoptosis. Furthermore, R428 sensitized cancer cells to multiple anti-cancer drugs-mediated cell death. Our results provide that inhibition of Axl could improve sensitivity to TRAIL through downregulation of c-FLIP and survivin expression in renal carcinoma cells. Taken together, Axl may be a tempting target to overcome TRAIL resistance.

Z-FL-COCHO, a cathepsin S inhibitor, enhances oxaliplatin-induced apoptosis through upregulation of Bim expression.

Inhibition of cathespsin S not only inhibits invasion and angiogenesis, but also induces apoptosis and autophagy in cancer cells. In present study, we revealed that pharmacological inhibitor [Z-FL-COCHO (ZFL)] of cathepsin S up-regulates pro-apoptotic protein Bim expression at the posttranslational levels. These effects were not associated with MAPKs and AMPK signal pathways. Interestingly, pretreatment with the chemical chaperones (TUDCA and PBA) and knockdown of protein phosphatase 2A (PP2A) markedly inhibited ZFL-induced Bim upregulation. ZFL enhances oxaliplatin-mediated apoptosis through ER stress-induced Bim upregulation in cancer cells. Collectively, our results suggest that inhibition of cathepsin S-induced Bim upregulation contribute to anti-cancer drug-induced apoptotic cell death in renal carcinoma Caki cells.

Angelicin potentiates TRAIL-induced apoptosis in renal carcinoma Caki cells through activation of caspase 3 and down-regulation of c-FLIP expression.

Preclinical Research & Development Angelicin is a furocoumarin derived from Psoralea corylifolia L. fruit that has anti-inflammatory and anti-tumor activity. In the present study, the effect of angelicin in enhancing tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptotic cell death was studied in Caki (renal carcinoma) cells. Angelicin alone and TRAIL alone had no effect on apoptosis, but in combination these compounds markedly induced apoptosis in the cancer cell lines while not inducing apoptosis in normal cells. The combination treatment induced accumulation of the sub-G1 population, DNA fragmentation, and activated caspase 3 activity in Caki cells, induced down-regulation of c-FLIP expression post-translationally, and over-expression of c-FLIP markedly blocked apoptosis induced by combined treatment with angelicin plus TRAIL. This study provides evidence that angelicin might be a TRAIL sensitizer.

Cepharanthine Enhances TRAIL-Mediated Apoptosis Through STAMBPL1-Mediated Downregulation of Survivin Expression in Renal Carcinoma Cells.

Cepharanthine (CEP) is a natural plant alkaloid, and has anti-inflammatory, antineoplastic, antioxidative and anticancer properties. In this study, we investigated whether CEP could sensitize renal carcinoma Caki cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. CEP alone and TRAIL alone had no effect on apoptosis. However, combined CEP and TRAIL treatment markedly enhanced apoptotic cell death in cancer cells, but not in normal cells. CEP induced downregulation of survivin and cellular-FLICE inhibitory protein (c-FLIP) expression at post-translational levels. Ectopic expression of survivin blocked apoptosis by combined treatment with CEP plus TRAIL, but not in c-FLIP overexpression. Interestingly, CEP induced survivin downregulation through downregulation of deubiquitin protein of STAM-binding protein-like 1 (STAMBPL1). Overexpression of STAMBPL1 markedly recovered CEP-mediated survivin downregulation. Taken together, our study suggests that CEP sensitizes TRAIL-mediated apoptosis through downregulation of survivin expression at the post-translational levels in renal carcinoma cells.

Corosolic Acid Induces Non-Apoptotic Cell Death through Generation of Lipid Reactive Oxygen Species Production in Human Renal Carcinoma Caki Cells.

Corosolic acid is one of the pentacyclic triterpenoids isolated from Lagerstroemia speciose and has been reported to exhibit anti-cancer and anti-proliferative activities in various cancer cells. In the present study, we investigated the molecular mechanisms of corosolic acid in cancer cell death. Corosolic acid induces a decrease of cell viability and an increase of cell cytotoxicity in human renal carcinoma Caki cells. Corosolic acid-induced cell death is not inhibited by apoptosis inhibitor (z-VAD-fmk, a pan-caspase inhibitor), necroptosis inhibitor (necrostatin-1), or ferroptosis inhibitors (ferrostatin-1 and deferoxamine (DFO)). Furthermore, corosolic acid significantly induces reactive oxygen species (ROS) levels, but antioxidants (N-acetyl-l-cysteine (NAC) and trolox) do not inhibit corosolic acid-induced cell death. Interestingly, corosolic acid induces lipid oxidation, and α-tocopherol markedly prevents corosolic acid-induced lipid peroxidation and cell death. Anti-chemotherapeutic effects of α-tocopherol are dependent on inhibition of lipid oxidation rather than inhibition of ROS production. In addition, corosolic acid induces non-apoptotic cell death in other renal cancer (ACHN and A498), breast cancer (MDA-MB231), and hepatocellular carcinoma (SK-Hep1 and Huh7) cells, and α-tocopherol markedly inhibits corosolic acid-induced cell death. Therefore, our results suggest that corosolic acid induces non-apoptotic cell death in cancer cells through the increase of lipid peroxidation.

Involvement of Up-Regulation of DR5 Expression and Down-Regulation of c-FLIP in Niclosamide-Mediated TRAIL Sensitization in Human Renal Carcinoma Caki Cells.

Niclosamide is used to treat intestinal parasite infections, as being an anthelmintic drug. Recently, several papers suggest the niclosamide inhibits multiple signaling pathways, which are highly activated and mutated in cancer. Here, niclosamide was evaluated for identifying strategies to overcome tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) resistance. Although niclosamide (100⁻200 nM) alone did not bring about cell death, combinations of niclosamide and TRAIL led to apoptotic cell death in carcinoma cells, but not in normal cells. Niclosamide markedly increased DR5 protein levels, including cell-surface DR5, and decreased c-FLIP protein levels. Down-regulation of DR5 by specific small interfering RNA (siRNA) and ectopic expression of c-FLIP markedly blocked niclosamide plus TRAIL-induced apoptosis. Our findings provide that niclosamide could overcome resistance to TRAIL through up-regulating DR5 on the cell surface and down-regulating c-FLIP in cancer cells. Taken together, niclosamide may be an attractive candidate to overcome TRAIL resistance.

HSP70 Acetylation Prevents Combined mTORC1/2 Inhibitor and Curcumin Treatment-Induced Apoptosis.

We previously reported that PP242 (dual inhibitor of mTORC1/2) plus curcumin induced apoptotic cell death through lysosomal membrane permeabilization (LMP)-mediated autophagy. However, the relationship between ER stress and apoptotic cell death by combined PP242 and curcumin treatment remains unknown. In the present study, we found that combined PP242 and curcumin treatment induced cytosolic Ca2+ release and ER stress. Interestingly, pretreatment with the chemical chaperones (TUDCA and 4-PBA) and knockdown of CHOP and ATF4 by siRNA did not abolish combined treatment-induced apoptosis in renal carcinoma cells. These results suggest that combined treatment with mTORC1/2 inhibitor and curcumin induces ER stress which is not essential for apoptotic cell death. Furthermore, overexpression of HSP70 significantly inhibited PP242 plus curcumin-induced LMP and apoptosis, but the protective effect was abolished by K77R mutation of acetylation site of HSP70. Taken together, our results reveal that regulation of HSP70 through K77 acetylation plays role in combined PP242 and curcumin treatment-induced apoptosis.

Garcinol Enhances TRAIL-Induced Apoptotic Cell Death through Up-Regulation of DR5 and Down-Regulation of c-FLIP Expression.

Garcinol is a polyisoprenylated benzophenone derived from the Garcinia indica fruit that possess potential therapeutic effects such as inhibition of inflammation and tumor expansion. Here, we investigated whether garcinol induces TRAIL sensitization in renal carcinoma cells. Single treatment with garcinol or TRAIL did not effect on apoptosis. However, combined treatment with garcinol plus TRAIL significantly induced apoptosis in renal carcinoma (Caki, ACHN and A498), lung carcinoma (A549), and hepatoma (SK-Hep1) cells. In contrast, garcinol plus TRAIL did not alter cell viability in normal cells. Garcinol plus TRAIL induced up-regulation of DR5 and down-regulation of c-FLIP expression at post-translational levels. Furthermore, knock-down of DR5 by siRNA and ectopic expression of c-FLIP blocked apoptotic cell death induced by garcinol plus TRAIL. Overall, our study provides evidence that garcinol can be exploited as a potential TRAIL sensitizer.

Maritoclax Enhances TRAIL-Induced Apoptosis via CHOP-Mediated Upregulation of DR5 and miR-708-Mediated Downregulation of cFLIP.

Maritoclax, an active constituent isolated from marine bacteria, has been known to induce Mcl-1 downregulation through proteasomal degradation. In this study, we investigated the sensitizing effect of maritoclax on tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in human renal carcinoma cells. We found that combined treatment with maritoclax and TRAIL markedly induced apoptosis in renal carcinoma (Caki, ACHN and A498), lung cancer (A549) and hepatocellular carcinoma (SK-Hep1) cells. The upregulation of death receptor 5 (DR5) and downregulation of cellular FLICE-inhibitory protein (cFLIP) were involved in maritoclax plus TRAIL-induced apoptosis. Maritoclax-induced DR5 upregulation was regulated by induction of C/EBP homologous protein (CHOP) expression. Interestingly, maritoclax induced cFLIP downregulation through the increased expression of miR-708. Ectopic expression of cFLIP prevented combined maritoclax and TRAIL-induced apoptosis. Taken together, maritoclax sensitized TRAIL-induced apoptosis through CHOP-mediated DR5 upregulation and miR-708-mediated cFLIP downregulation.

Volasertib Enhances Sensitivity to TRAIL in Renal Carcinoma Caki Cells through Downregulation of c-FLIP Expression.

Polo-like kinase 1 (PLK1) plays major roles in cell cycle control and DNA damage response. Therefore, PLK1 has been investigated as a target for cancer therapy. Volasertib is the second-in class dihydropteridinone derivate that is a specific PLK1 inhibitor. In this study, we examined that combining PLK1 inhibitor with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) would have an additive and synergistic effect on induction of apoptosis in cancer cells. We found that volasertib alone and TRAIL alone had no effect on apoptosis, but the combined treatment of volasertib and TRAIL markedly induced apoptosis in Caki (renal carcinoma), A498 (renal carcinoma) and A549 (lung carcinoma) cells, but not in normal cells (human skin fibroblast cells and mesangial cells). Combined treatment induced accumulation of sub-G1 phase, DNA fragmentation, cleavage of poly (ADP-ribose) polymerase (PARP) and activation of caspase 3 activity in Caki cells. Interestingly, combined treatment induced downregulation of cellular-FLICE-inhibitory protein (c-FLIP) expression and ectopic expression of c-FLIP markedly blocked combined treatment-induced apoptosis. Therefore, this study demonstrates that volasertib may sensitize TRAIL-induced apoptosis in Caki cells via downregulation of c-FLIP.

Cathepsin D promotes polarization of tumor-associated macrophages and metastasis through TGFBI-CCL20 signaling.

M2-like tumor-associated macrophages (TAMs) are risk factors for cancer progression and metastasis. However, the mechanisms underlying their polarization are still not fully understood. Although cathepsin D (Cat D) has been reported as a procarcinogenic factor, little is known about the functional role of Cat D in the tumor microenvironment (TME). This study aimed to explore the effect and molecular mechanisms of Cat D in the TME. Cat D knockout (KO) altered the cytokine secretion pattern and induced TAM reprogramming from the M2 to M1 subtype, thereby preventing epithelial-mesenchymal transition and tumor metastasis. Mechanistically, we identified transforming growth factor beta-induced protein (TGFBI) as a Cat D target protein that is specifically associated with TAM polarization. Elevated TGFBI expression in Cat D KO cancer cells resulted in a decline in M2-like TAM polarization. Our RNA-sequencing results indicated that the cancer cell-secreted chemokine CCL20 is a major secretory chemokine for Cat D-TGFBI-mediated TAM polarization. In contrast, Cat D overexpression accelerated TAM polarization into M2-like cells by suppressing TGFBI expression. In addition, the double Cat D and TGFBI KO rescued the inhibitory effects of Cat D KO on tumor metastasis by controlling TAM and T-cell activation. These findings indicated that Cat D contributes to cancer metastasis through TGFBI-mediated TAM reprogramming. Cat D deletion inhibits M2-like TAM polarization through TGFBI-mediated CCL20 expression, reprogramming the immunosuppressive TME. Our results open a potential new avenue for therapy focused on eliminating tumor metastasis.

Non-canonical deubiquitination of OTUB1 induces IFNγ-mediated cell cycle arrest via regulation of p27 stability.

The deubiquitinase OTUB1, implicated as a potential oncogene in various tumors, lacks clarity in its regulatory mechanism in tumor progression. Our study investigated the effects and underlying mechanisms of OTUB1 on the breast cancer cell cycle and proliferation in IFNγ stimulation. Loss of OTUB1 abrogated IFNγ-induced cell cycle arrest by regulating p27 protein expression, whereas OTUB1 overexpression significantly enhanced p27 expression even without IFNγ treatment. Tyr26 phosphorylation residue of OTUB1 directly bound to p27, modulating its post-translational expression. Furthermore, we identified crucial lysine residues (K134, K153, and K163) for p27 ubiquitination. Src downregulation reduced OTUB1 and p27 expression, suggesting that IFNγ-induced cell cycle arrest is mediated by the Src-OTUB1-p27 signaling pathway. Our findings highlight the pivotal role of OTUB1 in IFNγ-induced p27 expression and cell cycle arrest, offering therapeutic implications.

Cathepsin K inhibition induces Raptor destabilization and mitochondrial dysfunction via Syk/SHP2/Src/OTUB1 axis-mediated signaling.

The Raptor signaling pathway is a critical point of intervention in the invasion and progression of cancer. The non-receptor tyrosine kinase Src-mediated phosphorylation of OTUB1-Y26 plays a critical role in Raptor stabilization, whereas cathepsin K inhibitor (odanacatib; ODN) and knockdown (siRNA) induce Raptor destabilization. However, the mechanisms involved in cathepsin K inhibition-induced OTUB1-Y26 phosphorylation in Raptor stabilization have not been yet elucidated. This study showed that cathepsin K inhibition activates SHP2, a tyrosine phosphatase, that dephosphorylates OTUB1 and destabilizes Raptor, whereas SHP2 deletion and pharmacological inhibition increase OTUB1-Y26 phosphorylation and Raptor expression. SHP2 deletion also led to the inhibition of ODN-induced mitochondrial ROS, fusion, and dysfunction. Furthermore, cathepsin K inhibition phosphorylated spleen tyrosine kinase (Syk) at Y525 and Y526, resulting in the SHP2-mediated dephosphorylation of OTUB1-Y26. Collectively, our findings identified Syk not only as an upstream tyrosine kinase required for SHP2 activation but also showed a critical mechanism that regulates ODN-induced Raptor downregulation and mitochondrial dysfunction. In conclusion, Syk/SHP2/Src/OTUB1 axis-mediated signaling can act as a therapeutic target in cancer management.

Phosphorylation of OTUB1 at Tyr 26 stabilizes the mTORC1 component, Raptor.

Raptor plays a critical role in mTORC1 signaling. High expression of Raptor is associated with resistance of cancer cells to PI3K/mTOR inhibitors. Here, we found that OTUB1-stabilized Raptor in a non-canonical manner. Using biochemical assays, we found that the tyrosine 26 residue (Y26) of OTUB1 played a critical role in the interaction between OTUB1 and Raptor. Furthermore, non-receptor tyrosine kinases (Src and SRMS kinases) induced phosphorylation of OTUB1 at Y26, which stabilized Raptor. Interestingly, phosphorylation of OTUB1 at Y26 did not affect the stability of other OTUB1-targeted substrates. However, dephosphorylation of OTUB1 destabilized Raptor and sensitized cancer cells to anti-cancer drugs via mitochondrial reactive oxygen species-mediated mitochondrial dysfunction. Furthermore, we detected high levels of phospho-OTUB1 and Raptor in samples of patients with renal clear carcinoma. Our results suggested that regulation of OTUB1 phosphorylation may be an effective and selective therapeutic target for treating cancers via down-regulation of Raptor.