Research Progress in Function and Regulation of E3 Ubiquitin Ligase SMURF1

Smad ubiquitylation regulatory factor 1(Smurf1)is an important homologous member of E6-AP C-terminus type E3 ubiquitin ligase.Initially,Smurf1 was reportedly involved in the negative regulation of the bone morphogenesis protein(BMP)pathway.After further research,several studies have confirmed that Smurf1 is widely involved in various biological processes,such as bone homeostasis regulation,cell migration,apoptosis,and planar cell polarity.At the same time,recent studies have provided a deeper understanding of the regulatory mechanisms of Smurf1’s expression,activity,and substrate selectivity.In our review,a brief summary of recent important biological functions and regulatory mechanisms of E3 ubiquitin ligase Smurf1 is proposed.

Human RING finger protein ZNF645 is a novel testis-specific E3 ubiquitin ligase

A large number of testis-specific genes are involved in the complex process of mammalian spermatogenesis. Identification of these genes and their roles is important for understanding the mechanisms underlying spermatogenesis. Here we report on a novel human RING finger protein, ZNF645, which contains a C3HC4 RING finger domain, a C2H2 zinc-finger domain, and a proline-rich region, indicating that it has a structure similar to that of the c-Cbl-like protein Hakai. ZNF645 was exclusively expressed in normal human testicular tissue. Immunohistochemical analysis confirmed that ZNF645 protein was present in spermatocytes, round and elongated spermatids, and Leydig cells. Immunofluorescence staining of mature sperms further showed that the ZNF645 protein was localized over the postacrosomal perinuclear theca region and the entire length of sperm tail. An in vitro ubiquitination assay indicated that the RING finger domain of the ZNF645 protein had E3 ubiquitin ligase activity. Therefore, we suggest that ZNF645 might act as an E3 ubiquitin-protein ligase and play a role in human sperm production and quality control.

The E3 ubiquitin ligase seven in absentia homolog 1 may be a potential new therapeutic target for Parkinson's disease

In this study, we investigated the effect of an antibody against E3 ubiquitin ligase seven in absentia homolog 1（SIAH-1） in PC12 cells. 1-Methyl-4-phenylpyridinium（MPP＋） treatment increased α-synuclein, E1 and SIAH-1 protein levels in PC12 cells, and it reduced cell viability; however, there was no significant change in light chain 3 expression. Treatment with an SIAH-1 antibody decreased m RNA expression levels of α-synuclein, light chain 3 and SIAH-1, but increased E1 m RNA expression. It also increased cell viability. Combined treatment with MPP＋ and rapamycin reduced SIAH-1 and α-synuclein levels. Treatment with SIAH-1 antibody alone diminished α-synuclein immunoreactivity in PC12 cells, and reduced the colocalization of α-synuclein and light chain 3. These findings suggest that the SIAH-1 antibody reduces the monoubiquitination and aggregation of α-synuclein, promoting its degradation by the ubiquitin-proteasome pathway. Consequently, SIAH-1 may be a potential new therapeutic target for Parkinson’s disease.

Jianpi Decoction Combined with Medroxyprogesterone Acetate Alleviates Cancer Cachexia and Prevents Muscle Atrophy by Directly Inhibiting E3 Ubiquitin Ligase

ObjectiveTo provide comprehensive evidence for the anti-cancer cachexia effect of Jianpi Decoction(JP)and to explore its mechanism of anti-cancer cachexia.MethodsA mouse model of colon cancer(CT26)-induced cancer cachexia(CC)was used to investigate the anti-CC effect of JP combined with medroxyprogesterone acetate(MPA).Thirty-six mice were equally divided into 6 groups:normal control,CC,MPA(100 mg·kg^(−1)·d^(−1)),MPA+low-dose(20 mg·kg^(−1)·d^(−1))JP(L-JP),MPA+medium-dose(30 mg·kg^(−1)·d^(−1))JP(M-JP),and MPA+high-dose(40 mg·kg^(−1)·d^(−1))JP(H-JP)groups.After successful modeling,the mice were administered by gavage for 11 d.The body weight and tumor volume were measured and recorded every 2 d starting on the 8th day after implantation.The liver,heart,spleen,lung,kidney,tumor and gastrocnemius muscle of mice were collected and weighed.The pathological changes of the tumor was observed,and the cross-sectional area of the gastrocnemius muscle was calculated.The protein expressions of STAT3 and E3 ubiquitinase in the gastrocnemius muscle were measured by Western blot.In addition,an in vitro C2C12 myotube formation model was established to investigate the role of JP in hindering dexamethasone-induced muscle atrophy.In vitro experiments were divided into control,model,and JP serum groups.After 2-d administration,microscopic photographs were taken and myotube diameters were calculated.Western blot was performed to measure the protein expressions of STAT3 and E3 ubiquitinase.ResultsJP combined with MPA restored tumor-induced weight loss(P<0.05,vs.CC)and muscle fiber size(P<0.01,vs.CC).Mechanistically,JP reduced the expression of atrophy-related proteins MuRF1 and MAFbx in tumor-induced muscle atrophy in vivo(P<0.05,vs.CC).In addition,JP reduced the expression of atrophy-related proteins MuRF1 and MAFbx and p-STAT3 phosphorylation(P<0.05 or P<0.01 vs.model group)in C2C12 myotubes treated with dexamethasone in vitro.ConclusionsAdministration of JP combined with MPA restores tumor-induced cachexia conditions.In addition,the profound effect of JP combined with MPA on tumor-induced cachexia may be due to its inhibition of muscle proteolysis(E3 ubiquitinase system).

The role of E3 ubiquitin ligases in bone homeostasis and related diseases

The ubiquitin-proteasome system(UPS)dedicates to degrade intracellular proteins to modulate demic homeostasis and functions of organisms.These enzymatic cascades mark and modifies target proteins diversly through covalently binding ubiquitin molecules.In the UPS,E3 ubiquitin ligases are the crucial constituents by the advantage of recognizing and presenting proteins to proteasomes for proteolysis.As the major regulators of protein homeostasis,E3 ligases are indispensable to proper cell manners in diverse systems,and they are well described in physiological bone growth and bone metabolism.Pathologically,classic bone-related diseases such as metabolic bone diseases,arthritis,bone neoplasms and bone metastasis of the tumor,etc.,were also depicted in a UPS-dependent manner.Therefore,skeletal system is versatilely regulated by UPS and it is worthy to summarize the underlying mechanism.Furthermore,based on the current status of treatment,normal or pathological osteogenesis and tumorigenesis elaborated in this review highlight the clinical significance of UPS research.As a strategy possibly remedies the limitations of UPS treatment,emerging PROTAC was described comprehensively to illustrate its potential in clinical application.Altogether,the purpose of this review aims to provide more evidence for exploiting novel therapeutic strategies based on UPS for bone associated diseases.

ESCRT-I Component VPS23A Is Targeted by E3 Ubiquitin Ligase XBAT35 for Proteasome-Mediated Degradation in Modulating ABA Signaling

A myriad of abiotic stress responses in plants are controlled by abscisic acid(ABA)signaling.ABA receptors can be degraded by both the 26S proteasome pathway and vacuolar degradation pathway after processing via the endosomal sorting complex required for transport(ESCRT)proteins.Despite being essential for ABA signaling,the upstream regulators of ESCRTs remain unknown.Here,we report that the ESCRT-I component VPS23A is an unstable protein that is degraded via the ubiquitin-proteasome system(UPS).The UEV domain of VPS23A physically interacts with the two PSAP motifs of XBAT35,an E3 ubiquitin ligase,and this interaction results in the deposition of K48 polyubiquitin chains on VPS23A,marking it for degradation by 26S proteasomes.We showed that XBAT35 in plants is a positive regulator of ABA responses that acts via the VPS23A/PYL4 complex,specifically by accelerating VPS23A turnover and thereby increasing accumulation of the ABA receptor PYL4.This work deciphers how an ESCRT component is regulated in plants and deepens our understanding of plant stress responses by illustrating a mechanism whereby crosstalk between the UPS and endosome-vacuole-mediated degradation pathways controls ABA signaling.

The E3 ubiquitin ligase HUWE1 acts through the N-Myc-DLL1-NOTCH1 signaling axis to suppress glioblastoma progression

Background:Elucidation of the post-transcriptional modification has led to novel strategies to treat intractable tumors,especially glioblastoma(GBM).The ubiquitin-proteasome system(UPS)mediates a reversible,stringent and stepwise post-translational modification which is closely associated with malignant processes of GBM.To this end,developing novel therapeutic approaches to target the UPS may contribute to the treatment of this disease.This study aimed to screen the vital and aberrantly regulated component of the UPS in GBM.Based on the molecular identification,functional characterization,and mechanism investigation,we sought to elaborate a novel therapeutic strategy to target this vital factor to combat GBM.Methods:We combined glioma datasets and human patient samples to screen and identify aberrantly regulated E3 ubiquitin ligase.Multidimensional database analysis and molecular and functional experiments in vivo and in vitro were used to evaluate the roles of HECT,UBA and WWE domain-containing E3 ubiquitin ligase 1(HUWE1)in GBM.dCas9 synergistic activation mediator system and recombinant adeno-associated virus(rAAV)were used to endogenously overexpress full-length HUWE1 in vitro and in glioma orthotopic xenografts.Results:Low expression of HUWE1 was closely associated with worse prognosis of GBM patients.The ubiquitination and subsequent degradation of N-Myc mediated by HUWE1,leading to the inactivation of downstream Delta-like 1(DLL1)-NOTCH1 signaling pathways,inhibited the proliferation,invasion,and migration of GBM cells in vitro and in vivo.A rAAV dual-vector system for packaging and delivery of dCas9-VP64 was used to augment endogenous HUWE1 expression in vivo and showed an antitumor activity in glioma orthotopic xenografts.Conclusions:The E3 ubiquitin ligase HUWE1 acts through the N-Myc-DLL1-NOTCH1 signaling axis to suppress GBM progression.Antitumor activity of rAAV dual-vector delivering dCas9-HUWE1 system uncovers a promising therapeutic strategy for GBM.

The emerging roles of E3 ubiquitin ligases in ovarian cancer chemoresistance

Epithelial cancer of the ovary exhibits the highest mortality rate of all gynecological malignancies in women today,since the disease is often diagnosed in advanced stages.While the treatment of cancer with specific chemical agents or drugs is the favored treatment regimen,chemotherapy resistance greatly impedes successful ovarian cancer chemotherapy.Thus,chemoresistance becomes one of the most critical clinical issues confronted when treating patients with ovarian cancer.Convincing evidence hints that dysregulation of E3 ubiquitin ligases is a key factor in the development and maintenance of ovarian cancer chemoresistance.This review outlines recent advancement in our understanding of the emerging roles of E3 ubiquitin ligases in ovarian cancer chemoresistance.We also highlight currently available inhibitors targeting E3 ligase activities and discuss their potential for clinical applications in treating chemoresistant ovarian cancer patients.

Fine mapping of a major QTL qHYF_B06 for peanut yield

High yield is a major objective for peanut(Arachis hypogaea L.) breeding worldwide. However, fewer yield-related quantitative trait loci(QTL) have been reported in peanut than in other staple food crops such as rice(Oryza sativa), wheat(Triticum aestivum), and maize(Zea mays). This study aimed to identify stable major-effect QTL associated with pod yield per plant, hundred-pod weight for double-seeded pods,hundred-seed weight, shelling percentage, and pod number per plant, allowing us to predict candidate genes by means of transcriptome and genome sequencing. To this end, we used a population of recombinant inbred lines comprising 192 F9:11families derived from a JH6 × KX01-6 cross to construct a highresolution genetic map(1705.7 c M) consisting of 2273 polymorphic SNPs, with 0.75 c M(on average)between adjacent SNPs. We identified two high-confidence, yield-related QTL, qHYF_A08 and qHYF_B06, explaining 5.78%–31.40% of phenotypic variation and with LOD values of 5.10–24.48, in six environments. qHYF_A08 mainly explained the variation in shelling percentage, whereas qHYF_B06explained variation in hundred-pod weight and hundred-seed weight and accounted for 8.77%–31.40%of the variation in effective pod number per plant, pod number per plant, and shelling percentage. We narrowed down qHYF_B06 to an 890-kb interval using an advanced mapping population.Transcriptome and genome analyses revealed that only Arahy.129FS0 and Arahy.3R9A5K in the candidate mapping interval were differentially expressed between JH6 and KX01-6, with substantial structural variations in their promoter and coding regions. Genotypes of 208 peanut accessions determined using a diagnostic CAPS marker suggested that the two haplotypes of Arahy.3R9A5K were highly associated with hundred-seed weight and hundred-pod weight;this diagnostic CAPs marker could therefore be useful for selecting high-yielding lines during peanut breeding. Overall, our results provide valuable information for cloning alleles with favorable effects on peanut yield.

A novel missense mutation of the ubiquitin protein ligase E3A gene in a patient with Angelman syndrome

Background Angelman syndrome （AS） is a neurogenetic disorder caused by an expression defect of the maternally inherited copy of ubiquitin protein ligase E3A （UBE3A） gene from chromosome 15. Although the most common genetic defects include maternal deletions of chromosome 15q11-13, paternal uniparental disomy and imprinting defect, mutations in the UBE3A gene have been identified in approximately 10% of AS patients. Methods A Chinese girl of 28 months presented clinical manifestation of AS. Genetic diagnosis and molecular genetic defects were studied by methylation-specific PCR （MS-PCR） and linkage analysis by short tandem repeat （STR）. We further performed sequence analysis of all the coding exons and flanking sequences of the UBE3A gene. The novel mutation screening was also performed in 100 unrelated healthy individuals to exclude the possibility of identifying a polymorphism variation. Results The MS-PCR analysis of the patient showed biparental inheritance of chromosome 15 with a normal methylation pattern in the 15q11-q13 region. And STR analysis revealed that the patient also inherited biparental alleles for six microsatellites. A novel mutation, cDNA1199 C〉A （p.P400H）, in exon 9 of the maternal UBE3A gene, was identified in the patient. Meanwhile, the mutation was observed in the patient＇s mother who had a normal phenotype. Conclusions It is necessary to perform the UBE3A gene mutation analysis in non-deletion/non-UPD/non-ID patients with AS. The clinical picture of the patient is concordant with that observed in previously reported AS patients with UBE3A mutation.

Drosophila ubiquitin E3 ligase dSmurf is required for synapse remodeling and axon pruning by glia

Animal behaviors and higher-order functions rely on complex neural circuits built by synaptic connections （synapses） to deliver messages among different brain cells. As the major mediator in the nervous system, neurons communicate via synapses, which undergo constant structural remodeling with strict regulation.

Functional Characterization of the Apple RING E3 Ligase MdMIEL1 in Transgenic Arabidopsis

E3 ubiquitin ligases are involved in various physiological processes,and they play pivotal roles in growth and development.In this study,we identified a previously unknown gene in the apple fruit(Malus×domestica)and named it MdMIEL1.The MdMIEL1 gene encoded a protein that contained a zinc-finger domain at its N-terminus and a RING-finger motif at its C-terminus.To investigate MdMIEL1 functions,we generated transgenic Arabidopsis lines expressing the MdMIEL1 gene under the control of the Cauliflower mosaic virus 35S promoter.Interestingly,ectopic expression of MdMIEL1 in Arabidopsis produced multiple phenotypes,including early germination,early flowering and a lateral root number increase relative to wild-type plants.Further analysis indicated that MdMIEL1 regulated lateral root initiation by increasing auxin accumulation in the roots.In a word,these results suggest that,MdMIEL1 as a novel RING-finger ubiquitin ligase influences plant growth and development,and highlight that MdMIEL1 regulates lateral root growth.

Overexpressed NEDD8 as a potential therapeutic target in esophageal squamous cell carcinoma

Objective:The hyperactivated neddylation pathway plays an important role in tumorigenesis and is emerging as a promising anticancer target.We aimed to study whether NEDD8(neural precursor cell expressed,developmentally down-regulated 8)might serve as a therapeutic target in esophageal squamous cell carcinoma(ESCC).Methods:The clinical relevance of NEDD8 expression was evaluated by using The Cancer Genome Atlas(TCGA)database and tissue arrays.NEDD8-knockdown ESCC cells generated with the CRISPR/Cas9 system were used to explore the anticancer effects and mechanisms.Quantitative proteomic analysis was used to examine the variations in NEDD8 knockdown-induced biological pathways.The cell cycle and apoptosis were assessed with fluorescence activated cell sorting.A subcutaneous-transplantation mouse tumor model was established to investigate the anticancer potential of NEDD8 silencing in vivo.Results:NEDD8 was upregulated at both the mRNA and protein expression levels in ESCC,and NEDD8 overexpression was associated with poorer overall patient survival(mRNA level:P=0.028,protein level:P=0.026,log-rank test).Downregulation of NEDD8 significantly suppressed tumor growth both in vitro and in vivo.Quantitative proteomic analysis revealed that downregulation of NEDD8 induced cell cycle arrest,DNA damage,and apoptosis in ESCC cells.Mechanistic studies demonstrated that NEDD8 knockdown led to the accumulation of cullin-RING E3 ubiquitin ligases(CRLs)substrates through inactivation of CRLs,thus suppressing the malignant phenotype by inducing cell cycle arrest and apoptosis in ESCC.Rescue experiments demonstrated that the induction of apoptosis after NEDD8 silencing was attenuated by DR5 knockdown.Conclusions:Our study elucidated the anti-ESCC effects and underlying mechanisms of NEDD8 knockdown,and validated NEDD8 as a potential target for ESCC therapy.

The testis-specifically expressed gene Trim69 is not essential for fertility in mice

Protein ubiquitination is essential for diverse cellular functions including spermatogenesis.The tripartite motif(TRIM)family proteins,most of which have E3 ubiquitin ligase activity,are highly conserved in mammals.They are involved in important cellular processes such as embryonic development,immunity,and fertility.Our previous studies indicated that Trim69,a testis-specific expressed TRIM family gene,potentially participates in the spermatogenesis by mediating testicular cells apoptosis.In this study,we investigated the biological functions of Trim69 in male mice by established Trim69 knockout mice with CRISPR/Cas9 genomic editing technology.Here,we reported that the male Trim69 knockout mice had normal fertility.The adult knockout mice have shown that the appearance of testes,testis/body weight ratios,testicular histomorphology,and the number and quality of sperm were consistent with wild-type mice.These results indicated that the E3 ubiquitin ligase protein Trim69 was not essential for male mouse fertility,and it might be compensated by other TRIM family members such as Trim58 in Trim69-deficiency testis.This study would help to elucidate the functions of tripartite motif protein family and the regulation of spermatogenesis.

Ubiquitin ligase DTX3 empowers mutant p53 to promote ovarian cancer development

The deltex family protein DTX3 is believed to possess E3 ubiquitin ligase activity,as it contains a classic RING finger domain.However,its biological role and the underlying mechanism in cancer remain largely elusive.Here,we identified DTX3 as a novel mutant p53-interacting protein in ovarian carcinoma.Mechanistically,DTX3 mediated mutant p53 ubiqui-tination and stabilization by perturbing the MDM2-mutant p53 interaction,consequently leading to activation of diverse.mutant p53 target genes.Importantly,a positive correlation between the expression of DTX3 and mutant p53 target genes was further validated in ovarian carcinomas.Ectopic DTX3 promoted,while depletion of DTX3 suppressed,ovarian cancer cell proliferation and invasion.Remarkably,the pro-tumorigenic effect of DTX3 is dependent on mutant p53,because ablation of mutant p53 significantly impaired DTX3-induced gene expression and ovarian cancer cell growth and propagation.Furthermore,DTX3 elevated the expressi on of muta nt p53 target genes and boosted ovarian tumor growth in vivo.Fin ally,DTX3 was amplified and overexpressed in ovarian carci no mas,which is sign ificantly associated with unfavorable prognosis.Altogether,our findings unveil the oncogenic role of DTX3 in ovarian cancer development by bolstering mutant p53 activity.

Genome-wide identification of U-box gene family and expression analysis under abiotic stresses in Salvia miltiorrhiza

Plant U-box(PUB)E3 ubiquitin ligases play important roles in hormone signaling pathways and in response to different abiotic stresses,but little is known about U-box genes in Danshen(root of Salvia miltiorrhiza Bunge).Here,we identified and characterized 70 SmPUB genes based on its genome sequence.Phylogenetic analysis of U-box genes from S.miltiorrhiza and Arabidopsis suggested that they can be clustered into seven subgroups(I–VII).Typical U-box domains were found in all identified SmPUB genes through the analysis of conserved motifs.Moreover,qRT-PCR was applied to analyze the relative expression levels of U-box genes in S.miltiorrhiza roots and leaves under PEG-induced water deficit and salt stresses.Results revealed that the SmPUB genes exhibited stronger response to drought than to salt stress.To the best of our knowledge,this report is the first to perform genome-wide identification and analysis of the U-box gene family in S.miltiorrhiza,and the results provide valuable information for better understanding of the function of U-box in S.miltiorrhiza.

An alfalfa MYB-like transcriptional factor MsMYBH positively regulates alfalfa seedling drought resistance and undergoes MsWAV3-mediated degradation

Drought is a major threat to alfalfa(Medicago sativa L.)production.The discovery of important alfalfa genes regulating drought response will facilitate breeding for drought-resistant alfalfa cultivars.Here,we report a genome-wide association study of drought resistance in alfalfa.We identified and functionally characterized an MYB-like transcription factor gene(MsMYBH),which increases the drought resistance in alfalfa.Compared with the wild-types,the biomass and forage quality were enhanced in MsMYBH overexpressed plants.Combined RNA-seq,proteomics and chromatin immunoprecipitation analysis showed that MsMYBH can directly bind to the promoters of MsMCP1,MsMCP2,MsPRX1A and MsCARCAB to improve their expression.The outcomes of such interactions include better water balance,high photosynthetic efficiency and scavenge excess H_(2)O_(2)in response to drought.Furthermore,an E3 ubiquitin ligase(MsWAV3)was found to induce MsMYBH degradation under long-term drought,via the 26S proteasome pathway.Furthermore,variable-number tandem repeats in MsMYBH promoter were characterized among a collection of germplasms,and the variation is associated with promoter activity.Collectively,our findings shed light on the functions of MsMYBH and provide a pivotal gene that could be leveraged for breeding drought-resistant alfalfa.This discovery also offers new insights into the mechanisms of drought resistance in alfalfa.

Ubiquitination modification:critical regulation of IRF family stability and activity

Interferon regulatory factors(IRFs)play pivotal and critical roles in innate and adaptive immune responses;thus,precise and stringent regulation of the stability and activation of IRFs in physiological processes is necessary.The stability and activities of IRFs are directly or indirectly targeted by endogenous and exogenous proteins in an ubiquitin-dependent manner.However,few reviews have summarized how host E3 ligases/DUBs or viral proteins regulate IRF stability and activity.Additionally,with recent technological developments,details about the ubiquitination of IRFs have been continuously revealed.As knowledge of how these proteins function and interact with IRFs may facilitate a better understanding of the regulation of IRFs in immune responses or other biological processes,we summarized current studies on the direct ubiquitination of IRFs,with an emphasis on how these proteins interact with IRFs and affect their activities,which may provide exciting targets for drug development by regulating the functions of specific E3 ligases.

The Photomorphogenic Central Repressor COP1: Conservation and Functional Diversification during Evolution

Green plants on the earth have evolved intricate mechanisms to acclimatize to and utilize sunlight.In Arabidopsis,light signals are perceived by photoreceptors and transmitted through divergent but overlapping signaling networks to modulate plant photomorphogenic development.COP1(CONSTITUTIVE PHOTOMORPHOGENIC 1)was first cloned as a central repressor of photomorphogenesis in higher plants and has been extensively studied for over 30 years.It acts as a RING E3 ubiquitin ligase downstream of multiple photoreceptors to target key light-signaling regulators for degradation,primarily as part of large protein complexes.The mammalian counterpart of COP1 is a pluripotent regulator of tumorigenesis and metabolism.A great deal of information on COP1 has been derived from whole-genome sequencing and functional studies in lower green plants,which enables us to illustrate its evolutionary history.Here,we reviewthe current understanding about COP1,with a focus on the conservation and functional diversification of COP1 and its signaling partners in different taxonomic clades.

mTOR-targeted cancer therapy:great target but disappointing clinical outcomes,why?

The mammalian target of rapamycin(mTOR)critically regulates several essential biological functions,such as cell growth,metabolism,survival,and immune response by forming two important complexes,namely,mTOR complex 1(mTORC1)and complex 2(mTORC2).mTOR signaling is often dysregulated in cancers and has been considered an attractive cancer therapeutic target.Great efforts have been made to develop efficacious mTOR inhibitors,particularly mTOR kinase inhibitors,which suppress mTORC1 and mTORC2;however,major success has not been achieved.With the strong scientific rationale,the intriguing question is why cancers are insensitive or not responsive to mTOR-targeted cancer therapy in clinics.Beyond early findings on induced activation of PI3K/Akt,MEK/ERK,and Mnk/eIF4E survival signaling pathways that compromise the efficacy of rapalog-based cancer therapy,recent findings on the essential role of GSK3 in mediating cancer cell response to mTOR inhibitors and mTORC1 inhibition-induced upregulation of PD-L1 in cancer cells may provide some explanations.These new findings may also offer us the opportunity to rationally utilize mTOR inhibitors in cancer therapy.Further elucidation of the biology of complicated mTOR networks may bring us the hope to develop effective therapeutic strategies with mTOR inhibitors against cancer.