p21-activated kinase 4 counteracts PKA-dependent lipolysis by phosphorylating FABP4 and HSL.

Adipose tissue lipolysis is mediated by cAMP-protein kinase A (PKA)-dependent intracellular signalling. Here, we show that PKA targets p21-activated kinase 4 (PAK4), leading to its protein degradation. Adipose tissue-specific overexpression of PAK4 in mice attenuates lipolysis and exacerbates diet-induced obesity. Conversely, adipose tissue-specific knockout of Pak4 or the administration of a PAK4 inhibitor in mice ameliorates diet-induced obesity and insulin resistance while enhancing lipolysis. Pak4 knockout also increases energy expenditure and adipose tissue browning activity. Mechanistically, PAK4 directly phosphorylates fatty acid-binding protein 4 (FABP4) at T126 and hormone-sensitive lipase (HSL) at S565, impairing their interaction and thereby inhibiting lipolysis. Levels of PAK4 and the phosphorylation of FABP4-T126 and HSL-S565 are enhanced in the visceral fat of individuals with obesity compared to their lean counterparts. In summary, we have uncovered an important role for FABP4 phosphorylation in regulating adipose tissue lipolysis, and PAK4 inhibition may offer a therapeutic strategy for the treatment of obesity.

Identification of α-amanitin effector proteins in hepatocytes by limited proteolysis-coupled mass spectrometry.

The misuse of poisonous mushrooms containing amatoxins causes acute liver failure (ALF) in patients and is a cause of significant mortality. Although the toxic mechanisms of α-amanitin (α-AMA) and its interactions with RNA polymerase II (RNAP II) have been studied, α-AMA effector proteins that can interact with α-AMA in hepatocytes have not been systematically studied. Limited proteolysis-coupled mass spectrometry (LiP-MS) is an advanced technology that can quickly identify protein-ligand interactions based on global comparative proteomics. This study identified the α-AMA effector proteins found in human hepatocytes, following the detection of conformotypic peptides using LiP-MS coupled with tandem mass tag (TMT) technology. Proteins that are classified into protein processing in the endoplasmic reticulum and the ribosome during the KEGG pathway can be identified through affinity evaluation, according to α-AMA concentration-dependent LiP-MS and LiP-MS in hepatocytes derived from humans and mice, respectively. The possibility of interaction between α-AMA and proteins containing conformotypic peptides was evaluated through molecular docking studies. The results of this study suggest a novel path for α-AMA to induce hepatotoxicity through interactions with various proteins involved in protein synthesis, as well as with RNAP II.

p21-activated kinase 4 suppresses fatty acid ß-oxidation and ketogenesis by phosphorylating NCoR1.

PPARα corepressor NCoR1 is a key regulator of fatty acid ß-oxidation and ketogenesis. However, its regulatory mechanism is largely unknown. Here, we report that oncoprotein p21-activated kinase 4 (PAK4) is an NCoR1 kinase. Specifically, PAK4 phosphorylates NCoR1 at T1619/T2124, resulting in an increase in its nuclear localization and interaction with PPARα, thereby repressing the transcriptional activity of PPARα. We observe impaired ketogenesis and increases in PAK4 protein and NCoR1 phosphorylation levels in liver tissues of high fat diet-fed mice, NAFLD patients, and hepatocellular carcinoma patients. Forced overexpression of PAK4 in mice represses ketogenesis and thereby increases hepatic fat accumulation, whereas genetic ablation or pharmacological inhibition of PAK4 exhibites an opposite phenotype. Interestingly, PAK4 protein levels are significantly suppressed by fasting, largely through either cAMP/PKA- or Sirt1-mediated ubiquitination and proteasome degradation. In this way, our findings provide evidence for a PAK4-NCoR1/PPARα signaling pathway that regulates fatty acid ß-oxidation and ketogenesis.

Sesamin ameliorates lipotoxicity and lipid accumulation through the activation of the estrogen receptor alpha signaling pathway.

Nonalcoholic fatty liver disease (NAFLD) has been linked to fat accumulation in the liver and lipid metabolism imbalance. Sesamin, a lignan commonly found in sesame seed oil, possesses antioxidant, anti-inflammatory, and anticancer properties. However, the precise mechanisms by which sesamin prevents hepatic steatosis are not well understood. This study aimed to explore the molecular mechanisms by which sesamin may improve lipid metabolism dysregulation. A in vitro hepatic steatosis model was established by exposing HepG2 cells to palmitate sodium. The results showed that sesamin effectively mitigated lipotoxicity and reduced reactive oxygen species production. Additionally, sesamin suppressed lipid accumulation by regulating key factors involved in lipogenesis and lipolysis, such as fatty acid synthase (FASN), sterol regulatory element-binding protein 1c (SREBP-1c), forkhead box protein O-1, and adipose triglyceride lipase. Molecular docking results indicated that sesamin could bind to estrogen receptor α (ERα) and reduce FASN and SREBP-1c expression via the Ca2+/calmodulin-dependent protein kinase kinase ß (CaMKKß)/AMP-activated protein kinase (AMPK) signaling pathway. Sesamin attenuated palmitate-induced lipotoxicity and regulated hepatic lipid metabolism in HepG2 cells by activating the ERα/CaMKKß/AMPK signaling pathway. These findings suggest that sesamin can improve lipid metabolism disorders and is a promising candidate for treating hepatic steatosis.

Ethyl Gallate Isolated from Castanopsis cuspidata var. sieboldii Branches Inhibits Melanogenesis and Promotes Autophagy in B16F10 Cells.

The Castanopsis cuspidata var. sieboldii (CCS) plant grows predominantly in temperate regions of Asian countries, such as South Korea. Research on CCS has so far concentrated on the nutritional analysis, antioxidant activity, and anti-inflammation properties of its branches. However, the isolation of compounds and structural elucidation of effective single molecules remain unexplored, necessitating further exploration of CCS branches. Therefore, this study demonstrates the antioxidant and antimelanogenic activity of a single substance of ethyl gallate (EG) isolated from CCS branch extracts. Notably, the antimelanogenic (whitening) activity of EG extracted from CCS branches remains unexplored. Tyrosinase inhibition, kinetic enzyme assays, and molecular docking studies were conducted using mushroom tyrosinase in order to examine the antioxidant mechanism and antimelanin activity of EG in B16F10 melanoma cells. Nontoxic EG concentrations were found to be below 5 µg/mL. While EG significantly reduced the levels of whitening-associated proteins, p-CREB, and p-PKA, it dose-dependently inhibited the expression of TYR, TRP-1, TRP-2, and transcription factor (MITF). In addition, EG downregulated melanogenetic gene expression and activated autophagy signals. Therefore, EG extracted from CCS branches could serve as a novel functional cosmetic material with antimelanogenic and autophagy-enhancing activity.

Nomilin from Yuzu Seed Has In Vitro Antioxidant Activity and Downregulates Melanogenesis in B16F10 Melanoma Cells through the PKA/CREB Signaling Pathway.

Yuzu (Citrus junos) is a citrus plant native to Asian countries, including Korea, Japan, and China. Yuzu peel and seed contain abundant vitamin C, citric acid, and polyphenols. Although the antioxidative and antimelanogenic activities of other citrus fruits and yuzu extract have been reported, the tyrosinase inhibitory activity of the limonoid aglycone contained in yuzu seed extract is unknown. We separated yuzu seeds into the husk, shell, and meal and evaluated antioxidant activity of each. The limonoid glucoside fraction of the husk identified nomilin, a novel tyrosinase inhibitor. We performed tyrosinase inhibitory activity and noncompetitive inhibition assays and docking studies to determine nomilin binding sites. Furthermore, we evaluated the antioxidative mechanism and antimelanogenic activity of nomilin in B16F10 melanoma cells. The concentration of nomilin that did not show toxicity was <100 µg/mL. Nomilin suppressed protein expression of TYR, TRP-1, TRP-2, and microphthalmia-associated transcription factor (MITF) in a concentration-dependent manner. Nomilin significantly reduced the levels of p-CREB and p-PKA at the protein level and decreased the levels of skin-whitening-related factors MITF, tyrosinase, TRP-1, and TRP-2 at the mRNA level in a concentration-dependent manner. Thus, nomilin from yuzu seed husk can be used as a skin-whitening agent in cosmetics.

Inhibition of phospholipase D1 induces immunogenic cell death and potentiates cancer immunotherapy in colorectal cancer.

Phospholipase D (PLD) is a potential therapeutic target against cancer. However, the contribution of PLD inhibition to the antitumor response remains unknown. We developed a potent and selective PLD1 inhibitor based on computer-aided drug design. The inhibitor enhanced apoptosis in colorectal cancer (CRC) cells but not in normal colonic cells, and in vitro cardiotoxicity was not observed. The inhibitor downregulated the Wnt/ß-catenin signaling pathway and reduced the migration, invasion, and self-renewal capacity of CRC cells. In cancer, therapeutic engagement of immunogenic cell death (ICD) leads to more effective responses by eliciting the antitumor immunity of T cells. The CRC cells treated with the inhibitor showed hallmarks of ICD, including downregulation of "do not eat-me" signals (CD24, CD47, programmed cell death ligand 1 [PD-L1]), upregulation of "eat-me" signal (calreticulin), release of high-mobility group Box 1, and ATP. PLD1 inhibition subsequently enhanced the phagocytosis of cancer cells by macrophages through the surface expression of costimulatory molecules; as a result, the cancer cells were more susceptible to cytotoxic T-cell-mediated killing. Moreover, PLD1 inhibition attenuated colitis-associated CRC and orthotopically injected tumors, probably by controlling multiple pathways, including Wnt signaling, phagocytosis checkpoints, and immune signaling. Furthermore, combination therapy with a PLD1 inhibitor and an anti-PD-L1 antibody further enhanced tumor regression via immune activation in the tumor environment. Collectively, in this study, PLD1 was identified as a critical regulator of the tumor microenvironment in colorectal cancer, suggesting the potential of PLD1 inhibitors for cancer immunotherapy based on ICD and immune activation. PLD1 inhibitors may act as promising immune modulators in antitumor treatment via ICD.

Puerarin attenuates hepatic steatosis via G-protein-coupled estrogen receptor-mediated calcium and SIRT1 signaling pathways.

Puerarin, the major bioactive ingredient isolated from the root of Pueraria lobata (Willd.), attenuates body weight gain and reduces lipid levels in high-fat diet-induced obese mice; however, the underlying mechanism responsible for regulating lipid metabolism remains unclear. This study investigated the molecular mechanism(s) underlying the role of puerarin in regulating lipogenesis and lipolysis in human HepG2 cells. In this study, puerarin strongly inhibited the expression of fatty acid synthase (FASN) and sterol regulatory element binding protein 1c (SREBP-1c). Moreover, puerarin significantly induced the expression of adipose triglyceride lipase (ATGL), which is responsible for triacylglycerol hydrolase activity in cells. Puerarin enhanced 5' AMP-activated protein kinase (AMPK) activity, which is a central regulator of hepatic lipid metabolism. Furthermore, this AMPK activation could be mediated by sirtuin 1 (SIRT1) and calcium signaling pathways involved in G protein-coupled estrogen receptor (GPER) signaling. GPER blockage significantly reversed the effect of puerarin on lipid accumulation and the related signaling pathways. Docking studies showed that puerarin could bind in the GPER in a similar manner as GPER agonist G1. Our results suggest that puerarin can improve hepatic steatosis by activating GPER; it's signaling cascade sequentially induced calcium and SIRT1 signaling pathways. Thus, puerarin may be a potential therapeutic agent for the treatment of non-alcoholic fatty liver disease.

Identification of Pyridinyltriazine Derivatives as Potent panFGFR Inhibitors against Gatekeeper Mutants for Overcoming Drug Resistance.

Although FGFR inhibitors hold promise in treating various cancers, resistance to the FGFR inhibitors caused by acquired secondary mutations has emerged. To discover novel FGFR inhibitors capable of inhibiting FGFR mutations, including gatekeeper mutations, we designed and synthesized several new pyridinyltriazine derivatives. A structure-activity relationship (SAR) study led to the identification of 17a as a highly potent panFGFR inhibitor against wild-type and mutant FGFRs. Notably, 17a is superior to infigratinib in terms of kinase-inhibitory and cellular activities, especially against V555M-FGFR3. Molecular dynamics simulations provide a clear understanding of why pyridinyltraizine derivative 17a possesses activity against V555M-FGFR3. Moreover, 17a significantly suppresses proliferation of cancer cells harboring FGFR mutations via FGFR signaling blockade, cell cycle arrest, and apoptosis. Furthermore, 17a and 17b exhibited remarkable efficacies in TEL-V555M-FGFR3 Ba/F3 xenograft mouse model and 17a is more efficacious than infigratinib. This study provides new insight into the design of novel FGFR inhibitors that are active against FGFR mutants.

p21-activated kinase 4 inhibition protects against liver ischemia/reperfusion injury: Role of nuclear factor erythroid 2-related factor 2 phosphorylation.

BACKGROUND AND AIMS: p21-activated kinase 4 (PAK4), an oncogenic protein, has emerged as a promising target for anticancer drug development. Its role in oxidative stress conditions, however, remains elusive. We investigated the effects of PAK4 signaling on hepatic ischemia/reperfusion (I/R) injury. APPROACH AND RESULTS: Hepatocyte- and myeloid-specific Pak4 knockout (KO) mice and their littermate controls were subjected to a partial hepatic I/R (HIR) injury. We manipulated the catalytic activity of PAK4, either through genetic engineering (gene knockout, overexpression of wild-type [WT] or dominant-negative kinase) or pharmacological inhibitor, coupled with a readout of nuclear factor erythroid 2-related factor 2 (Nrf2) activity, to test the potential function of PAK4 on HIR injury. PAK4 expression was markedly up-regulated in liver during HIR injury in mice and humans. Deletion of PAK4 in hepatocytes, but not in myeloid cells, ameliorated liver damages, as demonstrated in the decrease in hepatocellular necrosis and inflammatory responses. Conversely, the forced expression of WT PAK4 aggravated the pathological changes. PAK4 directly phosphorylated Nrf2 at T369, and it led to its nuclear export and proteasomal degradation, all of which impaired antioxidant responses in hepatocytes. Nrf2 silencing in liver abolished the protective effects of PAK4 deficiency. A PAK4 inhibitor protected mice from HIR injury. CONCLUSIONS: PAK4 phosphorylates Nrf2 and suppresses its transcriptional activity. Genetic or pharmacological suppression of PAK4 alleviates HIR injury. Thus, PAK4 inhibition may represent a promising intervention against I/R-induced liver injury.

Novel Macrocyclic Peptidomimetics Targeting the Polo-Box Domain of Polo-Like Kinase 1.

The polo-box domain (PBD) of Plk1 is a promising target for cancer therapeutics. We designed and synthesized novel phosphorylated macrocyclic peptidomimetics targeting PBD based on acyclic phosphopeptide PMQSpTPL. The inhibitory activities of 16e on Plk1-PBD is >30-fold higher than those of PMQSpTPL. Both 16a and 16e possess excellent selectivity for Plk1-PBD over Plk2/3-PBD. Analysis of the cocrystal structure of Plk1-PBD in complex with 16a reveals that the 3-(trifluoromethyl)benzoyl group in 16a interacts with Arg516 through a π-stacking interaction. This π-stacking interaction, which has not been reported previously, provides insight into the design of novel and potent Plk1-PBD inhibitors. Furthermore, 16h, a PEGlyated macrocyclic phosphopeptide derivative, induces Plk1 delocalization and mitotic failure in HeLa cells. Also, the number of phospho-H3-positive cells in a zebrafish embryo increases in proportion to the amount of 16a. Collectively, the novel macrocyclic peptidomimetics should serve as valuable templates for the design of potent and novel Plk1-PBD inhibitors.

Combination of Type I and Type II MET Tyrosine Kinase Inhibitors as Therapeutic Approach to Prevent Resistance.

MET-targeted therapies are clinically effective in MET-amplified and MET exon 14 deletion mutant (METex14) non-small cell lung cancers (NSCLCs), but their efficacy is limited by the development of drug resistance. Structurally distinct MET tyrosine kinase inhibitors (TKIs) (type I/II) have been developed or are under clinical evaluation, which may overcome MET-mediated drug resistance mechanisms. In this study, we assess secondary MET mutations likely to emerge in response to treatment with single-agent or combinations of type I/type II MET TKIs using TPR-MET transformed Ba/F3 cell mutagenesis assays. We found that these inhibitors gave rise to distinct secondary MET mutant profiles. However, a combination of type I/II TKI inhibitors (capmatinib and merestinib) yielded no resistant clones in vitro The combination of capmatinib/merestinib was evaluated in vivo and led to a significant reduction in tumor outgrowth compared with either MET inhibitor alone. Our findings demonstrate in vitro and in vivo that a simultaneous treatment with a type I and type II MET TKI may be a clinically viable approach to delay and/or diminish the emergence of on target MET-mediated drug-resistance mutations.

Identification of Thiazolo[5,4-b]pyridine Derivatives as c-KIT Inhibitors for Overcoming Imatinib Resistance.

c-KIT is a promising therapeutic target against gastrointestinal stromal tumor (GIST). In order to identify novel c-KIT inhibitors capable of overcoming imatinib resistance, we synthesized 31 novel thiazolo[5,4-b]pyridine derivatives and performed SAR studies. We observed that, among these substances, 6r is capable of inhibiting significantly c-KIT and suppressing substantially proliferation of GIST-T1 cancer cells. It is of note that 6r is potent against a c-KIT V560G/D816V double mutant resistant to imatinib. Compared with sunitinib, 6r possesses higher differential cytotoxicity on c-KIT D816V Ba/F3 cells relative to parental Ba/F3 cells. In addition, kinase panel profiling reveals that 6r has reasonable kinase selectivity. It was found that 6r remarkably attenuates proliferation of cancer cells via blockade of c-KIT downstream signaling, and induction of apoptosis and cell cycle arrest. Furthermore, 6r notably suppresses migration and invasion, as well as anchorage-independent growth of GIST-T1 cells. This study provides useful SAR information for the design of novel c-KIT inhibitors overcoming imatinib-resistance.

Acacetin Inhibits the Growth of STAT3-Activated DU145 Prostate Cancer Cells by Directly Binding to Signal Transducer and Activator of Transcription 3 (STAT3).

Signal transducer and activator of transcription 3 (STAT3) plays a critical role in the formation and growth of human cancer. Therefore, STAT3 is a therapeutic target for cancer drug discovery. Acacetin, a flavone present in various plants, inhibits constitutive and inducible STAT3 activation in STAT3-activated DU145 prostate cancer cells. Acacetin inhibits STAT3 activity by directly binding to STAT3, which we confirmed by a pull-down assay with a biotinylated compound and two level-free methods, namely, a drug affinity responsive target stability (DARTS) experiment and a cellular thermal shift assay (CETSA). Acacetin inhibits STAT3 phosphorylation at the tyrosine 705 residue and nuclear translocation in DU145 cells, which leads to the downregulation of STAT3 target genes. Acacetin then induces apoptosis in a time-dependent manner. Interestingly, acacetin induces the production of reactive oxygen species (ROS) that are not involved in the acacetin-induced inhibition of STAT3 activation because the suppressed p-STAT3 level is not rescued by treatment with GSH or NAC, which are general ROS inhibitors. We also found that acacetin inhibits tumor growth in xenografted nude mice. These results suggest that acacetin, as a STAT3 inhibitor, could be a possible drug candidate for targeting STAT3 for the treatment of cancer in humans.

Identification of Thieno[3,2-d]pyrimidine Derivatives as Dual Inhibitors of Focal Adhesion Kinase and FMS-like Tyrosine Kinase 3.

Focal adhesion kinase (FAK) is overexpressed in highly invasive and metastatic cancers. To identify novel FAK inhibitors, we designed and synthesized various thieno[3,2-d]pyrimidine derivatives. An intensive structure-activity relationship (SAR) study led to the identification of 26 as a lead. Moreover, 26, a multitargeted kinase inhibitor, possesses excellent potencies against FLT3 mutants as well as FAK. Gratifyingly, 26 remarkably inhibits recalcitrant FLT3 mutants, including F691L, that cause drug resistance. Importantly, 26 is superior to PF-562271 in terms of apoptosis induction, anchorage-independent growth inhibition, and tumor burden reduction in the MDA-MB-231 xenograft mouse model. Also, 26 causes regression of tumor growth in the MV4-11 xenograft mouse model, indicating that it could be effective against acute myeloid leukemia (AML). Finally, in an orthotopic mouse model using MDA-MB-231, 26 remarkably prevents metastasis of orthotopic tumors to lymph nodes. Taken together, the results indicate that 26 possesses potential therapeutic value against highly invasive cancers and relapsed AML.

A Novel Protein-Protein Interaction between RSK3 and IκBα and a New Binding Inhibitor That Suppresses Breast Cancer Tumorigenesis.

Multiple cancer-related biological processes are mediated by protein-protein interactions (PPIs). Through interactions with a variety of factors, members of the ribosomal S6 kinase (RSK) family play roles in cell cycle progression and cell proliferation. In particular, RSK3 contributes to cancer viability, but the underlying mechanisms remain unknown. We performed a kinase library screen to find IκBα PPI binding partners and identified RSK3 as a novel IκBα binding partner using a cell-based distribution assay. In addition, we discovered a new PPI inhibitor using mammalian two-hybrid (MTH) analysis. We assessed the antitumor effects of the new inhibitor using cell proliferation and colony formation assays and monitored the rate of cell death by FACS apoptosis assay. IκBα is phosphorylated by the active form of the RSK3 kinase. A small-molecule inhibitor that targets the RSK3/IκBα complex exhibited antitumor activity in breast cancer cells and increased their rate of apoptosis. RSK3 phosphorylation and RSK3/IκBα complex formation might be functionally important in breast tumorigenesis. The RSK3/IκBα-specific binding inhibitor identified in this study represents a lead compound for the development of new anticancer drugs.

X-ray Crystal Structure-Guided Design and Optimization of 7H-Pyrrolo[2,3-d]pyrimidine-5-carbonitrile Scaffold as a Potent and Orally Active Monopolar Spindle 1 Inhibitor.

Triple-negative breast cancer (TNBC) is an aggressive breast-cancer subtype associated with poor prognosis and high relapse rates. Monopolar spindle 1 kinase (MPS1) is an apical dual-specificity protein kinase that is over-expressed in TNBC. We herein report a highly selective MPS1 inhibitor based on a 7H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile scaffold. Our lead optimization was guided by key X-ray crystal structure analysis. In vivo evaluation of candidate (9) is shown to effectively mitigate human TNBC cell proliferation.

Novel Quercetin Derivative of 3,7-Dioleylquercetin Shows Less Toxicity and Highly Potent Tyrosinase Inhibition Activity.

Quercetin is a well-known plant flavonol and antioxidant; however, there has been some debate regarding the efficacy and safety of native quercetin as a skin-whitening agent via tyrosinase inhibition. Several researchers have synthesized quercetin derivatives as low-toxicity antioxidants and whitening agents. However, no suitable quercetin derivatives have been reported to date. In this study, a novel quercetin derivative was synthesized by the SN2 reaction using quercetin and oleyl bromide. The relationship between the structures and activities of quercetin derivatives as anti-melanogenic agents was assessed using in vitro enzyme kinetics, molecular docking, and quenching studies; cell line experiments; and in vivo zebrafish model studies. Novel 3,7-dioleylquercetin (OQ) exhibited a low cytotoxic concentration level at >100 µg/mL (125 µM), which is five times less toxic than native quercetin. The inhibition mechanism showed that OQ is a competitive inhibitor, similar to native quercetin. Expression of tyrosinase, tyrosinase-related protein 1 (TRP-1) and tyrosinase-related protein 2 (TRP-2), and microphthalmia-associated transcription factor was inhibited in B16F10 melanoma cell lines. mRNA transcription levels of tyrosinase, TRP-1, and TRP-2 decreased in a dose-dependent manner. Melanin formation was confirmed in the zebrafish model using quercetin derivatives. Therefore, OQ might be a valuable asset for the development of novel skin-whitening agents.

Dutomycin Induces Autophagy and Apoptosis by Targeting the Serine Protease Inhibitor SERPINB6.

Autophagy plays an important role in maintaining tumor cell progression and survival in response to metabolic stress. Thus, the regulation of autophagy can be used as a strategy for anticancer therapy. Here, we report dutomycin (DTM) as a novel autophagy enhancer that eventually induces apoptosis due to excessive autophagy. Also, human serine protease inhibitor B6 (SERPINB6) was identified as a target protein of DTM, and its novel function which is involved in autophagy was studied for the first time. We show that DTM directly binds SERPINB6 and then activates intracellular serine proteases, resulting in autophagy induction. Inhibitory effects of DTM on the function of SERPINB6 were confirmed through enzyme- and cell-based approaches, and SERPINB6 was validated as a target protein using siRNA-mediated knockdown and an overexpression test. In a zebrafish xenograft model, DTM showed a significant decrease in tumor area. Furthermore, the present findings will be expected to contribute to the expansion of novel basic knowledge about the correlation of cancer and autophagy by promoting active further research on SERPINB6, which was not previously considered the subject of cancer biology.