NAMPT-Driven M2 Polarization of Tumor-Associated Macrophages Leads to an Immunosuppressive Microenvironment in Colorectal Cancer.

Nicotinamide phosphoribosyltransferase (NAMPT) is a metabolic enzyme with key roles in inflammation. Previous studies have examined the consequences of its upregulated expression in cancer cells themselves, but studies are limited with respect to its role in the other cells within the tumor microenvironment (TME) during colorectal cancer (CRC) progression. Using single-cell RNA sequencing (scRNA-seq) data, it is founded that NAMPT is highly expressed in SPP1+ tumor-associated macrophages (TAMs), a unique subset of TAMs associated with immunosuppressive activity. A NAMPThigh gene signature in SPP1+ TAMs correlated with worse prognostic outcomes in CRC patients. The effect of Nampt deletion in the myeloid compartment of mice during CRC development is explored. NAMPT deficiency in macrophages resulted in HIF-1α destabilization, leading to reduction in M2-like TAM polarization. NAMPT deficiency caused significant decreases in the efferocytosis activity of macrophages, which enhanced STING signaling and the induction of type I IFN-response genes. Expression of these genes contributed to anti-tumoral immunity via potentiation of cytotoxic T cell activity in the TME. Overall, these findings suggest that NAMPT-initiated TAM-specific genes can be useful in predicting poor CRC patient outcomes; strategies aimed at targeting NAMPT may provide a promising therapeutic approach for building an immunostimulatory TME in CRC progression.

Akt enhances the vulnerability of cancer cells to VCP/p97 inhibition-mediated paraptosis.

Valosin-containing protein (VCP)/p97, an AAA+ ATPase critical for maintaining proteostasis, emerges as a promising target for cancer therapy. This study reveals that targeting VCP selectively eliminates breast cancer cells while sparing non-transformed cells by inducing paraptosis, a non-apoptotic cell death mechanism characterized by endoplasmic reticulum and mitochondria dilation. Intriguingly, oncogenic HRas sensitizes non-transformed cells to VCP inhibition-mediated paraptosis. The susceptibility of cancer cells to VCP inhibition is attributed to the non-attenuation and recovery of protein synthesis under proteotoxic stress. Mechanistically, mTORC2/Akt activation and eIF3d-dependent translation contribute to translational rebound and amplification of proteotoxic stress. Furthermore, the ATF4/DDIT4 axis augments VCP inhibition-mediated paraptosis by activating Akt. Given that hyperactive Akt counteracts chemotherapeutic-induced apoptosis, VCP inhibition presents a promising therapeutic avenue to exploit Akt-associated vulnerabilities in cancer cells by triggering paraptosis while safeguarding normal cells.

SRSF7 downregulation induces cellular senescence through generation of MDM2 variants.

Alternative splicing (AS) enables a pre-mRNA to generate different functional protein variants. The change in AS has been reported as an emerging contributor to cellular senescence and aging. However, it remains to be elucidated which senescent AS variants are generated in and regulate senescence. Here, we observed commonly down-regulated SRSF7 in senescent cells, using publicly available RNA-seq datasets of several in vitro senescence models. We further confirmed SRSF7 deregulation from our previous microarray datasets of time-series replicative senescence (RS) and oxidative stress-induced senescence (OSIS) of human diploid fibroblast (HDF). We validated the time-course changes of SRSF mRNA and protein levels, developing both RS and OSIS. SRSF knockdown in HDF was enough to induce senescence, accompanied by p53 protein stabilization and MDM2 variants formation. Interestingly, expression of MDM2 variants showed similar patterns of p53 expression in both RS and OSIS. Next, we identified MDM2-C as a key functional AS variant generated specifically by SRSF7 depletion. Finally, we validated that MDM2-C overexpression induced senescence of HDF. These results indicate that SRSF7 down-regulation plays a key role in p53-mediated senescence by regulating AS of MDM2, a key negative regulator of p53, implying its critical involvement in the entry into cell senescence.

Mid-old cells are a potential target for anti-aging interventions in the elderly.

The biological process of aging is thought to result in part from accumulation of senescent cells in organs. However, the present study identified a subset of fibroblasts and smooth muscle cells which are the major constituents of organ stroma neither proliferative nor senescent in tissues of the elderly, which we termed "mid-old status" cells. Upregulation of pro-inflammatory genes (IL1B and SAA1) and downregulation of anti-inflammatory genes (SLIT2 and CXCL12) were detected in mid-old cells. In the stroma, SAA1 promotes development of the inflammatory microenvironment via upregulation of MMP9, which decreases the stability of epithelial cells present on the basement membrane, decreasing epithelial cell function. Remarkably, the microenvironmental change and the functional decline of mid-old cells could be reversed by a young cell-originated protein, SLIT2. Our data identify functional reversion of mid-old cells as a potential method to prevent or ameliorate aspects of aging-related tissue dysfunction.

Mitoribosomal Deregulation Drives Senescence via TPP1-Mediated Telomere Deprotection.

While mitochondrial bioenergetic deregulation has long been implicated in cellular senescence, its mechanistic involvement remains unclear. By leveraging diverse mitochondria-related gene expression profiles derived from two different cellular senescence models of human diploid fibroblasts, we found that the expression of mitoribosomal proteins (MRPs) was generally decreased during the early-to-middle transition prior to the exhibition of noticeable SA-ß-gal activity. Suppressed expression patterns of the identified senescence-associated MRP signatures (SA-MRPs) were validated in aged human cells and rat and mouse skin tissues and in aging mouse fibroblasts at single-cell resolution. TIN2- and POT1-interaction protein (TPP1) was concurrently suppressed, which induced senescence, accompanied by telomere DNA damage. Lastly, we show that SA-MRP deregulation could be a potential upstream regulator of TPP1 suppression. Our results indicate that mitoribosomal deregulation could represent an early event initiating mitochondrial dysfunction and serve as a primary driver of cellular senescence and an upstream regulator of shelterin-mediated telomere deprotection.

NAMPT mitigates colitis severity by supporting redox-sensitive activation of phagocytosis in inflammatory macrophages.

Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme in the nicotinamide adenine dinucleotide (NAD+) salvage pathway and plays a crucial role in the maintenance of the NAD+ pool during inflammation. Considering that macrophages are essential for tissue homeostasis and inflammation, we sought to examine the functional impact of NAMPT in inflammatory macrophages, particularly in the context of inflammatory bowel disease (IBD). In this study, we show that mice with NAMPT deletion within the myeloid compartment (Namptf/fLysMCre+/-, Nampt mKO) have more pronounced colitis with lower survival rates, as well as numerous uncleared apoptotic corpses within the mucosal layer. Nampt-deficient macrophages exhibit reduced phagocytic activity due to insufficient NAD+ abundance, which is required to produce NADPH for the oxidative burst. Nicotinamide mononucleotide (NMN) treatment rescues NADPH levels in Nampt mKO macrophages and sustains superoxide generation via NADPH oxidase. Consequently, Nampt mKO mice fail to clear dead cells during tissue repair, leading to substantially prolonged chronic colitis. Moreover, systemic administration of NMN, to supply NAD+, effectively suppresses the disease severity of DSS-induced colitis. Collectively, our findings suggest that activation of the NAMPT-dependent NAD+ biosynthetic pathway, via NMN administration, is a potential therapeutic strategy for managing inflammatory diseases.

MRPS31 loss is a key driver of mitochondrial deregulation and hepatocellular carcinoma aggressiveness.

Deregulated mitochondrial energetics is a metabolic hallmark of cancer cells. However, the causative mechanism of the bioenergetic deregulation is not clear. In this study, we show that somatic copy number alteration (SCNA) of mitoribosomal protein (MRP) genes is a key mechanism of bioenergetic deregulation in hepatocellular carcinoma (HCC). Association analysis between the genomic and transcriptomic profiles of 82 MRPs using The Cancer Genome Atlas-Liver HCC database identified eight key SCNA-dependent MRPs: MRPS31, MRPL10, MRPL21, MRPL15, MRPL13, MRPL55, and DAP3. MRPS31 was the only downregulated MRP harboring a DNA copy number (DCN) loss. MRPS31 loss was associated specifically with the DCN losses of many genes on chromosome 13q. Survival analysis revealed a unique dependency of HCC on the MRPS31 deficiency, showing poor clinical outcome. Subclass prediction analysis using several public classifiers indicated that MRPS31 loss is linked to aggressive HCC phenotypes. By employing hepatoma cell lines with SCNA-dependent MRPS31 expression (JHH5, HepG2, Hep3B, and SNU449), we demonstrated that MRPS31 deficiency is the key mechanism, disturbing the whole mitoribosome assembly. MRPS31 suppression enhanced hepatoma cell invasiveness by augmenting MMP7 and COL1A1 expression. Unlike the action of MMP7 on extracellular matrix destruction, COL1A1 modulated invasiveness via the ZEB1-mediated epithelial-to-mesenchymal transition. Finally, MRPS31 expression further stratified the high COL1A1/DDR1-expressing HCC groups into high and low overall survival, indicating that MRPS31 loss is a promising prognostic marker. SIGNIFICANCE: Our results provide new mechanistic insight for mitochondrial deregulation in HCC and present MRPS31 as a novel biomarker of HCC malignancy.

Global spliceosome activity regulates entry into cellular senescence.

Cellular senescence is a state of permanent growth arrest that can ultimately contribute to aging. Senescence can be induced by various stressors and is associated with a myriad of cellular functions and phenotypic markers. Alternative splicing is emerging as a critical contributor to senescence and aging. However, it is unclear how the composition and function of the spliceosome are involved in senescence. Here, using replicative and oxidative stress-induced senescence models in primary human fibroblasts, we report a common shift in the expression of 58 spliceosomal genes at the pre-senescence stage, prior to the detection of senescence-associated ß-galactosidase (SA-ß-gal) activity. Spliceosomal perturbation, induced by pharmacologic and genetic inhibition of splicesomal genes, triggered cells to enter senescence, suggesting a key role as a gatekeeper. Association analysis of transcription factors based on the 58 splicesomal genes revealed Sp1 as a key regulator of senescence entry. Indeed, Sp1 depletion suppressed the expression of downstream spliceosomal genes (HNRNPA3, SRSF7, and SRSF4) and effectively induced senescence. These results indicate that spliceosomal gene sets, rather than a single spliceosomal gene, regulate the early transition into senescence prior to SA-ß-gal expression. Furthermore, our study provides a spliceosome signature that may be used as an early senescence marker.

Mitochondrial Respiratory Defect Enhances Hepatoma Cell Invasiveness via STAT3/NFE2L1/STX12 Axis.

Mitochondrial respiratory defects have been implicated in cancer progression and metastasis, but how they control tumor cell aggressiveness remains unclear. Here, we demonstrate that a mitochondrial respiratory defect induces nuclear factor-erythroid 2 like 1 (NFE2L1) expression at the transcriptional level via reactive oxygen species (ROS)-mediated STAT3 activation. We identified syntaxin 12 (STX12) as an effective downstream target of NFE2L1 by performing cDNA microarray analysis after the overexpression and depletion of NFE2L1 in hepatoma cells. Bioinformatics analysis of The Cancer Genome Atlas Liver Hepatocellular carcinoma (TCGA-LIHC) open database (n = 371) also revealed a significant positive association (r = 0.3, p = 2.49 × 10-9) between NFE2L1 and STX12 expression. We further demonstrated that STX12 is upregulated through the ROS/STAT3/NFE2L1 axis and is a key downstream effector of NFE2L1 in modulating hepatoma cell invasiveness. In addition, gene enrichment analysis of TCGA-LIHC also showed that epithelial-mesenchymal transition (EMT)-related core genes are significantly upregulated in tumors co-expressing NFE2L1 and STX12. The positive association between NFE2L1 and STX12 expression was validated by immunohistochemistry of the hepatocellular carcinoma tissue array. Finally, higher EMT gene enrichment and worse overall survival (p = 0.043) were observed in the NFE2L1 and STX12 co-expression group with mitochondrial defect, as indicated by low NDUFA9 expression. Collectively, our results indicate that NFE2L1 is a key mitochondrial retrograde signaling-mediated primary gene product enhancing hepatoma cell invasiveness via STX12 expression and promoting liver cancer progression.

Association of TIM-3 expression with glucose metabolism in Jurkat T cells.

BACKGROUND: T cell activation is associated with increase in glycolysis and glutaminolysis. T cell immunoglobulin and mucin domain containing protein-3 (TIM-3), a T cell surface molecule, downregulates T cell activation and leads to insufficient immunity in cancer and chronic infection. TIM-3 regulates T cell activation possibly through alterations in metabolism; however, the relationship between TIM-3 expression and T cell metabolic changes has not been well studied. RESULTS: We investigated the association between TIM-3 expression and metabolic changes by analyzing glucose metabolism, glutamine metabolism, and mitochondrial function in TIM-3 overexpressing or knockout Jurkat T cell lines relative to their control cell lines. Glucose uptake and consumption, and lactate release were downregulated by TIM-3 expression but upregulated by TIM-3 knockout. Concomitantly, the expression of the glucose transporter, Glut1, but not Glut2, 3, or 4 was altered by TIM-3 expression. However, TIM-3 expression alone could not account for the change in glutamine consumption, glutamate release, and mitochondrial mass, ROS production or membrane potential in these cell lines. CONCLUSION: Our results show the association of TIM-3 expression with T cell glucose metabolism. These results are significant in chronic infections and cancers where it is necessary to control TIM-3 expressing T cells.

Mitoribosome Defect in Hepatocellular Carcinoma Promotes an Aggressive Phenotype with Suppressed Immune Reaction.

Mitochondrial ribosomes (mitoribosomes), the specialized translational machinery for mitochondrial genes, exclusively encode the subunits of the oxidative phosphorylation (OXPHOS) system. Although OXPHOS dysfunctions are associated with hepatic disorders including hepatocellular carcinoma (HCC), their underlying mechanisms remain poorly elucidated. In this study, we aimed to investigate the effects of mitoribosome defects on OXPHOS and HCC progression. By generating a gene signature from HCC transcriptome data, we developed a scoring system, i.e., mitoribosome defect score (MDS), which represents the degree of mitoribosomal defects in cancers. The MDS showed close associations with the clinical outcomes of patients with HCC and with gene functions such as oxidative phosphorylation, cell-cycle activation, and epithelial-mesenchymal transition. By analyzing immune profiles, we observed that mitoribosomal defects are also associated with immunosuppression and evasion. Taken together, our results provide new insights into the roles of mitoribosome defects in HCC progression.

Lactic acidosis caused by repressed lactate dehydrogenase subunit B expression down-regulates mitochondrial oxidative phosphorylation via the pyruvate dehydrogenase (PDH)-PDH kinase axis.

Aerobic glycolysis and mitochondrial dysfunction are key metabolic features of cancer cells, but their interplay during cancer development remains unclear. We previously reported that human hepatoma cells with mitochondrial defects exhibit down-regulated lactate dehydrogenase subunit B (LDHB) expression. Here, using several molecular and biochemical assays and informatics analyses, we investigated how LDHB suppression regulates mitochondrial respiratory activity and contributes to liver cancer progression. We found that transcriptional LDHB down-regulation is an upstream event during suppressed oxidative phosphorylation. We also observed that LDHB knockdown increases inhibitory phosphorylation of pyruvate dehydrogenase (PDH) via lactate-mediated PDH kinase (PDK) activation and thereby attenuates oxidative phosphorylation activity. Interestingly, monocarboxylate transporter 1 was the major lactate transporter in hepatoma cells, and its expression was essential for PDH phosphorylation by modulating intracellular lactate levels. Finally, bioinformatics analysis of the hepatocellular carcinoma cohort from The Cancer Genome Atlas revealed that a low LDHB/LDHA ratio is statistically significantly associated with poor prognostic outcomes. A low ratio was also associated with a significant enrichment in glycolysis genes and negatively correlated with PDK1 and 2 expression, supporting a close link between LDHB suppression and the PDK-PDH axis. These results suggest that LDHB suppression is a key mechanism that enhances glycolysis and is critically involved in the maintenance and propagation of mitochondrial dysfunction via lactate release in liver cancer progression.

Increased nicotinamide adenine dinucleotide pool promotes colon cancer progression by suppressing reactive oxygen species level.

Nicotinamide adenine dinucleotide (NAD) exists in an oxidized form (NAD+ ) and a reduced form (NADH). NAD+ plays crucial roles in cancer metabolism, including in cellular signaling, energy production and redox regulation. However, it remains unclear whether NAD(H) pool size (NAD+ and NADH) could be used as biomarker for colon cancer progression. Here, we showed that the NAD(H) pool size and NAD+ /NADH ratio both increased during colorectal cancer (CRC) progression due to activation of the NAD+ salvage pathway mediated by nicotinamide phosphoribosyltransferase (NAMPT). The NAMPT expression was upregulated in adenoma and adenocarcinoma tissues from CRC patients. The NADH fluorescence intensity measured by two-photon excitation fluorescence (TPEF) microscopy was consistently increased in CRC cell lines, azoxymethane/dextran sodium sulfate (AOM/DSS)-induced CRC tissues and tumor tissues from CRC patients. The increases in the NAD(H) pool inhibited the accumulation of excessive reactive oxygen species (ROS) levels and FK866, a specific inhibitor of NAMPT, treatment decreased the CRC nodule size by increasing ROS levels in AOM/DSS mice. Collectively, our results suggest that NAMPT-mediated upregulation of the NAD(H) pool protects cancer cells against detrimental oxidative stress and that detecting NADH fluorescence by TPEF microscopy could be a potential method for monitoring CRC progression.

Metabolic features and regulation in cell senescence.

Organismal aging is accompanied by a host of progressive metabolic alterations and an accumulation of senescent cells, along with functional decline and the appearance of multiple diseases. This implies that the metabolic features of cell senescence may contribute to the organism's metabolic changes and be closely linked to age-associated diseases, especially metabolic syndromes. However, there is no clear understanding of senescent metabolic characteristics. Here, we review key metabolic features and regulators of cellular senescence, focusing on mitochondrial dysfunction and anabolic deregulation, and their link to other senescence phenotypes and aging. We further discuss the mechanistic involvement of the metabolic regulators mTOR, AMPK, and GSK3, proposing them as key metabolic switches for modulating senescence. [BMB Reports 2019; 52(1): 5-12].

NNMT depletion contributes to liver cancer cell survival by enhancing autophagy under nutrient starvation.

Nicotinamide N-methyl transferase (NNMT) transfers a methyl group from S-adenosyl-L-methionine (SAM) to nicotinamide (NAM), producing 1-methylnicotinamide (1MNA). NNMT has been implicated in several cancer types and recently in metabolism, but its role in autophagy regulation has not yet been investigated. In this study, we determined that NNMT negatively regulated autophagy at the stage of ULK1 activation through protein phosphatase 2A (PP2A) activity. Specifically, NNMT knockdown increased PP2A methylation and subsequently enhanced phosphatase activity. Consequent p-ULK1 (S638) dephosphorylation derepressed ULK1 activity, resulting in autophagy induction. Accordingly, NNMT downregulation rescued tumor cells under nutrient deficiency in vivo, which was alleviated by ULK1 inhibitor treatment. In summary, our results suggest a novel mechanism by which tumor cells protect themselves against nutrient deprivation through NNMT suppression to accelerate autophagy.

Pyrrolidine dithiocarbamate reverses Bcl-xL-mediated apoptotic resistance to doxorubicin by inducing paraptosis.

Elevated Bcl-xL expression in cancer cells contributes to doxorubicin (DOX) resistance, leading to failure in chemotherapy. In addition, the clinical use of high-dose doxorubicin (DOX) in cancer therapy has been limited by issues with cardiotoxicity and hepatotoxicity. Here, we show that co-treatment with pyrrolidine dithiocarbamate (PDTC) attenuates DOX-induced apoptosis in Chang-L liver cells and human hepatocytes, but overcomes DOX resistance in Bcl-xL-overexpressing Chang-L cells and several hepatocellular carcinoma (HCC) cell lines with high Bcl-xL expression. Additionally, combined treatment with DOX and PDTC markedly retarded tumor growth in a Huh-7 HCC cell xenograft tumor model, compared to either mono-treatment. These results suggest that DOX/PDTC co-treatment may provide a safe and effective therapeutic strategy against malignant hepatoma cells with Bcl-xL-mediated apoptotic defects. We also found that induction of paraptosis, a cell death mode that is accompanied by dilation of the endoplasmic reticulum and mitochondria, is involved in this anti-cancer effect of DOX/PDTC. The intracellular glutathione levels were reduced in Bcl-xL-overexpressing Chang-L cells treated with DOX/PDTC, and DOX/PDTC-induced paraptosis was effectively blocked by pretreatment with thiol-antioxidants, but not by non-thiol antioxidants. Collectively, our results suggest that disruption of thiol homeostasis may critically contribute to DOX/PDTC-induced paraptosis in Bcl-xL-overexpressing cells.

Lactate-mediated mitoribosomal defects impair mitochondrial oxidative phosphorylation and promote hepatoma cell invasiveness.

Impaired mitochondrial oxidative phosphorylation (OXPHOS) capacity, accompanied by enhanced glycolysis, is a key metabolic feature of cancer cells, but its underlying mechanism remains unclear. Previously, we reported that human hepatoma cells that harbor OXPHOS defects exhibit high tumor cell invasiveness via elevated claudin-1 (CLN1). In the present study, we show that OXPHOS-defective hepatoma cells (SNU354 and SNU423 cell lines) exhibit reduced expression of mitochondrial ribosomal protein L13 (MRPL13), a mitochondrial ribosome (mitoribosome) subunit, suggesting a ribosomal defect. Specific inhibition of mitoribosomal translation by doxycycline, chloramphenicol, or siRNA-mediated MRPL13 knockdown decreased mitochondrial protein expression, reduced oxygen consumption rate, and increased CLN1-mediated tumor cell invasiveness in SNU387 cells, which have active mitochondria. Interestingly, we also found that exogenous lactate treatment suppressed MRPL13 expression and oxygen consumption rate and induced CLN1 expression. A bioinformatic analysis of the open RNA-Seq database from The Cancer Genome Atlas (TCGA) liver hepatocellular carcinoma (LIHC) cohort revealed a significant negative correlation between MRPL13 and CLN1 expression. Moreover, in patients with low MRPL13 expression, two oxidative metabolic indicators, pyruvate dehydrogenase B expression and the ratio of lactate dehydrogenase type B to type A, significantly and negatively correlated with CLN1 expression, indicating that the combination of elevated glycolysis and deficient MRPL13 activity was closely linked to CLN1-mediated tumor activity in LIHC. These results suggest that OXPHOS defects may be initiated and propagated by lactate-mediated mitoribosomal deficiencies and that these deficiencies are critically involved in LIHC development.

The Ubiquitin-like with PHD and Ring Finger Domains 1 (UHRF1)/DNA Methyltransferase 1 (DNMT1) Axis Is a Primary Regulator of Cell Senescence.

As senescence develops, cells sequentially acquire diverse senescent phenotypes along with simultaneous multistage gene reprogramming. It remains unclear what acts as the key regulator of the collective changes in gene expression at initiation of senescent reprogramming. Here we analyzed time series gene expression profiles obtained in two different senescence models in human diploid fibroblasts: replicative senescence and H2O2-induced senescence. Our results demonstrate that suppression of DNA methyltransferase 1 (DNMT1)-mediated DNA methylation activity was an initial event prior to the display of senescent phenotypes. We identified seven DNMT1-interacting proteins, ubiquitin-like with PHD and ring finger domains 1 (UHRF1), EZH2, CHEK1, SUV39H1, CBX5, PARP1, and HELLS (also known as LSH (lymphoid-specific helicase) 1), as being commonly down-regulated at the same time point as DNMT1 in both senescence models. Knockdown experiments revealed that, among the DNMT1-interacting proteins, only UHRF1 knockdown suppressed DNMT1 transcription. However, UHRF1 overexpression alone did not induce DNMT1 expression, indicating that UHRF1 was essential but not sufficient for DNMT1 transcription. Although UHRF1 knockdown effectively induced senescence, this was significantly attenuated by DNMT1 overexpression, clearly implicating the UHRF1/DNMT1 axis in senescence. Bioinformatics analysis further identified WNT5A as a downstream effector of UHRF1/DNMT1-mediated senescence. Senescence-associated hypomethylation was found at base pairs -1569 to -1363 from the transcription start site of the WNT5A gene in senescent human diploid fibroblasts. As expected, WNT5A overexpression induced senescent phenotypes. Overall, our results indicate that decreased UHRF1 expression is a key initial event in the suppression of DNMT1-mediated DNA methylation and in the consequent induction of senescence via increasing WNT5A expression.

Inhibition of Cathepsin S Induces Mitochondrial ROS That Sensitizes TRAIL-Mediated Apoptosis Through p53-Mediated Downregulation of Bcl-2 and c-FLIP.

AIMS: Cathepsin S is highly expressed in various cancer cells, and it has protumoral effects, including promotion of migration, invasion, and neovascularization. In this study, we show that inhibition of cathepsin S could sensitize cancer cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis. RESULTS: An inhibitor of cathepsin S (Z-FL-COCHO; ZFL) markedly induced apoptosis in human renal cancer cells treated with TRAIL. In contrast, combined treatment with ZFL and TRAIL had no effect on normal cells. ZFL downregulated Bcl-2 expression at the transcriptional level in a p53-dependent manner, and overexpression of Bcl-2 also markedly blocked apoptosis induced by combined treatment with ZFL and TRAIL. In addition, ZFL induced downregulation of c-FLIP, and overexpression of c-FLIP blocked the apoptosis induced by ZFL plus TRAIL. Moreover, ZFL increased the expression of Cbl, an E3 ligase of c-FLIP, in a p53-dependent manner, and knockdown of Cbl markedly prevented c-FLIP downregulation and the apoptosis induced by ZFL plus TRAIL. Interestingly, ZFL induced p53 expression via production of mitochondrial reactive oxygen species (ROS). We also demonstrated that downregulation of cathepsin S by small interfering RNA sensitized TRAIL-mediated apoptosis in Caki cells. INNOVATION: These results reveal the importance of cathepsin S on resistance against TRAIL, and inhibition of cathepsin S activity plays a crucial role in TRAIL-mediated cell death of cancer cells. CONCLUSION: Our results indicated that inhibition of cathepsin S stimulates TRAIL-induced apoptosis through downregulation of Bcl-2 and Cbl-mediated c-FLIP by ROS-mediated p53 expression. Antioxid. Redox Signal. 27, 215-233.