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p62/SQSTM1 (sequestosome-1) is a key protein involved in multiple cellular bioprocesses including autophagy, nutrient sensing, cell growth, cell death, and survival. Therefore, it is implicated in human diseases such as obesity and cancer. Here, we show that the CUL5–ASB6 complex is a ubiquitin E3 ligase complex mediating p62 ubiquitination and degradation. Depletion of CUL5 or ASB6 induced p62 accumulation, and overexpression of ASB6 promoted ubiquitination and degradation of p62. Functionally, ASB6 overexpression can inhibit the proliferation of MEF and hepatocellular carcinoma cells by reducing p62 protein level, and impair the occurrence of autophagy. Overall, our study identified a new molecular mechanism regulating p62 stability, which may provide additional insights for understanding the delicate control of p62 and cell proliferation–autophagy control in physiological and pathological settings.

Gastric cancer is the third leading cause of cancer-related death worldwide. The regulatory mechanisms underlying gastric cancer cell proliferation are largely unclear. Here, we show that the transcription factor GFI1 is associated with advanced clinical gastric cancer progression and promoted gastric cancer cell proliferation partially through inhibition of gastrokine-2 (GKN2) transcription. GFI1 was a degrading substrate of FBXW7, whose loss was observed in gastric cancer. Mechanistically, GSK3β-mediated GFI1 S94/S98 phosphorylation triggered its interaction with FBXW7, resulting in SCFFBXW7-mediated ubiquitination and degradation. A nondegradable GFI1 S94A/S98A mutant was more potent in driving gastric cancer cell proliferation and tumorigenesis than wild-type GFI1. Overall, this study reveals the oncogenic role of GFI1 in gastric cancer and provides mechanistic insights into the tumor suppressor function of FBXW7. Significance: These findings demonstrate the oncogenic role of the transcription factor GFI1 and the tumor suppressive function of FBXW7 in gastric cancer.

Summary: Drug resistance is a significant hindrance to effective cancer treatment. Although resistance mechanisms of epidermal growth factor receptor (EGFR) mutant cancer cells to lethal EGFR tyrosine kinase inhibitors (TKI) treatment have been investigated intensively, how cancer cells orchestrate adaptive response under sublethal drug challenge remains largely unknown. Here, we find that 2-h sublethal TKI treatment elicits a transient drug-tolerant state in EGFR mutant lung cancer cells. Continuous sublethal treatment reinforces this tolerance and eventually establishes long-term TKI resistance. This adaptive process involves H3K9 demethylation-mediated upregulation of branched-chain amino acid aminotransferase 1 (BCAT1) and subsequent metabolic reprogramming, which promotes TKI resistance through attenuating reactive oxygen species (ROS) accumulation. Combination treatment with TKI- and ROS-inducing reagents overcomes this drug resistance in preclinical mouse models. Clinical information analyses support the correlation of BCAT1 expression with the EGFR TKI response. Our findings reveal the importance of BCAT1-engaged metabolism reprogramming in TKI resistance in lung cancer. : How cancer cells, with strong plasticity, orchestrate their adaptive response under sublethal drug exposure remains largely unknown. Wang et al. show that sublethal tyrosine kinase inhibitor (TKI) treatment elicits drug resistance in EGFR-mutant lung cancer cells through H3K9 demethylation-mediated reprogramming of branched-chain amino acid (BCAA) metabolism. Keywords: lung cancer, EGFR tyrosine kinase inhibitors, drug resistance, metabolic reprogramming, branched-chain amino acids, BCAT1

Hepatitis B Virus (HBV) constitutes a major threat to global public health. Current understanding of HBV-host interaction is yet limited. Here, ribosome profiling, quantitative mass spectrometry and RNA-sequencing were conducted on a recently established HBV replication system, through which we identified multiomic differentially expressed genes (DEGs) that HBV orchestrated to remodel host proteostasis networks. Our multiomics interrogation revealed that HBV induced significant changes in both transcription and translation of 35 canonical genes including PPP1R15A, PGAM5 and SIRT6, as well as the expression of at least 15 non-canonical open reading frames (ncORFs) including ncPON2 and ncGRWD1, thus revealing an extra coding potential of human genome. Overexpression of these five genes but not the enzymatically deficient SIRT6 mutants suppressed HBV replication while knockdown of SIRT6 had opposite effect. Furthermore, the expression of SIRT6 was down-regulated in patients, cells or animal models of HBV infection. Mechanistic study further indicated that SIRT6 directly binds to mini-chromosome and deacetylates histone H3 lysine 9 (H3K9ac) and histone H3 lysine 56 (H3K56ac), and chemical activation of endogenous SIRT6 with MDL800 suppressed HBV infection in vitro and in vivo. By generating the first multiomics landscape of host-HBV interaction, our work is thus opening a new avenue to facilitate therapeutic development against HBV infection.

Toll-like receptors (TLRs) are a family of proteins that recognize pathogen associated molecular patterns (PAMPs). Their primary function is to activate innate immune responses while also involved in facilitating adaptive immune responses. Different TLRs exert distinct functions by activating varied immune cascades. Several TLRs are being pursued as cancer drug targets. We discovered a novel, highly potent and selective small molecule TLR8 agonist DN052. DN052 exhibited strong in vitro cellular activity with EC50 at 6.7 nM and was highly selective for TLR8 over other TLRs including TLR4, 7 and 9. The selectivity profile distinguished DN052 from all other TLR agonists currently in clinical development. DN052 displayed excellent in vitro ADMET and in vivo PK profiles. DN052 potently inhibited tumor growth as a single agent. Moreover, combination of DN052 with the immune checkpoint inhibitor, selected targeted therapeutics or chemotherapeutic drugs further enhanced efficacy of single agents. Mechanistically, treatment with DN052 resulted in strong induction of pro-inflammatory cytokines in ex vivo human PBMC assay and in vivo monkey study. GLP toxicity studies in rats and monkeys demonstrated favorable safety profile. This led to the advancement of DN052 into phase I clinical trials.

Cushing’s disease results from corticotroph adenomas of the pituitary that hypersecrete adrenocorticotropin (ACTH), leading to excess glucocorticoid and hypercortisolism. Mutations of the deubiquitinase gene USP8 occur in 35–62% of corticotroph adenomas. However, the major driver mutations in USP8 wild-type tumors remain elusive. Here, we report recurrent mutations in the deubiquitinase gene USP48 (predominantly encoding p.M415I or p.M415V; 21/91 subjects) and BRAF (encoding p.V600E; 15/91 subjects) in corticotroph adenomas with wild-type USP8. Similar to USP8 mutants, both USP48 and BRAF mutants enhance the promoter activity and transcription of the gene encoding proopiomelanocortin (POMC), which is the precursor of ACTH, providing a potential mechanism for ACTH overproduction in corticotroph adenomas. Moreover, primary corticotroph tumor cells harboring BRAF V600E are sensitive to the BRAF inhibitor vemurafenib. Our study thus contributes to the understanding of the molecular mechanism of the pathogenesis of corticotroph adenoma and informs therapeutic targets for this disease., In this study the authors report USP48 and BRAF are frequently mutated in USP8 wild-type corticotroph adenomas, and cause Cushing’s disease mainly through promoting the promoter activity of POMC. Inhibition of BRAF may be a promising therapeutic strategy for the treatment of patients with BRAF-mutated corticotroph adenomas.

Background & Aims Intrahepatic cholangiocarcinoma (ICC) is the second-most lethal primary liver cancer. Little is known about intratumoral heterogeneity (ITH) and its impact on ICC progression. We aimed to investigate the ITH of ICC in the hope of helping to develop new therapeutic strategies. Methods We obtained 69 spatially distinct regions from six operable ICCs. Patient-derived primary cancer cells (PDPCs) were established for each region, followed by whole-exome sequencing (WES) and multi-level validation. Results We observed widespread ITH for both somatic mutations and clonal architecture, shaped by multiple mechanisms, like clonal “illusion”, parallel evolution and chromosome instability. A median of 60.3% of mutations were heterogeneous, among which 85% of the driver mutations were located on the branches of tumor phylogenetic trees. Many truncal and clonal driver mutations occurred in tumor suppressor genes, such as TP53, SMARCB1 and PBRM1 that are involved in DNA repair and chromatin-remodeling. Genome doubling occurred in most cases (5/6) after the accumulation of truncal mutations and was shared by all intratumoral sub-regions. In all cases, ongoing chromosomal instability is evident throughout the evolutionary trajectory of ICC. The recurrence of ICC1239 provided evidence to support the polyclonal metastatic seeding in ICC. The change of mutation landscape and internal diversity among subclones during metastasis, such as the loss of chemoresistance mediator, can be used for new treatment strategies. Targeted therapy against truncal alterations, such as IDH1, JAK1, and KRAS mutations and EGFR amplification, was developed in 5/6 patients. Conclusions Integrated investigations of spatial ITH and clonal evolution may provide an important molecular foundation for enhanced understanding of tumorigenesis and progression in ICC. Lay summary We applied multiregional whole-exome sequencing to investigate the evolution of intrahepatic cholangiocarcinoma (ICC). The results revealed that many factors, such as parallel evolution and chromosome instability, may participate and promote the branch diversity of ICC. Interestingly, in one patient with primary and recurrent metastatic tumors, we found evidence of polyclonal metastatic seeding, indicating that symbiotic communities of multiple clones existed and were maintained during metastasis. More realistically, some truncal alterations, such as IDH1, JAK1, and KRAS mutations and EGFR amplification, could be promising treatment targets in patients with ICC.

Phosphoglycerate kinase 1 (PGK1) is an important enzyme in the metabolic glycolysis pathway. In this study, we observed a significant overexpression of PGK1 in liver cancer tissues and a negative correlation between PGK1 expression and liver cancer patient survival. Furthermore, depletion of PGK1 dramatically reduced cancer cell proliferation and tumorigenesis, indicating an oncogenic role of PGK1 in liver cancer progression. Moreover, we identified acetylation at the K323 site of PGK1 as an important regulatory mechanism for promoting its enzymatic activity and cancer cell metabolism. And we further characterized P300/cyclic adenosine monophosphate response element binding protein-binding protein-associated factor (PCAF) and Sirtuin 7 as the enzymes regulating K323 acetylation from both directions in liver cancer cells. Conclusion These findings demonstrate a novel regulation of PGK1 as well as its important role in liver cancer progression. (Hepatology 2017;65:515-528).

SUMMARYDrug resistance is a significant hindrance to effective cancer treatment. Although resistance mechanisms of epidermal growth factor receptor (EGFR)-mutant cancer cells to lethal EGFR tyrosine kinase inhibitors (TKI) treatment have been investigated intensively, how cancer cells orchestrate adaptive response under sublethal drug challenge remains largely unknown. Here we find that 2-hour sublethal TKI treatment elicits a transient drug-tolerant state in EGFR-mutant lung cancer cells. Continuous sublethal treatment reinforces this tolerance and eventually establishes long-term TKI resistance. This adaptive process involves H3K9 demethylation-mediated epigenetic upregulation of branched-chain amino acid aminotransferase 1 (BCAT1) and subsequent metabolic reprogramming, which promotes TKI resistance through attenuating reactive oxygen species (ROS) accumulation. Combinational treatment with TKI and ROS-inducing reagents overcomes this drug resistance in preclinical mouse models. Clinical information analyses support the correlation of BCAT1 expression with EGFR TKI response. Collectively, our findings reveal the importance of epigenetically regulated BCAT1-engaged metabolism reprogramming in TKI resistance in lung cancer.HIGHLIGHTSSublethal EGFR TKI treatment induces transient drug-tolerant state and long-term resistance in EGFR-mutant lung cancer cellsEpigenetically regulated BCAT1-mediated metabolic reprogramming orchestrates EGFR TKI-induced drug resistanceCombinational treatment with TKI and ROS-inducing agents overcomes the drug resistance induced by EGFR TKI treatment

In the version of this Article originally published, the labels for Rictor and mTOR in the whole cell lysate (WCL) blots were swapped in Fig. 3b and the mTOR blot was placed upside down. Unprocessed blots of mTOR were also missing from Supplementary Fig. 9. The corrected Figs are shown below. In addition, control blots for the mTOR antibody (Cell Signalling Technology #2972) were also missing. These are now provided below, as Fig. 9, and show that the lower band is likely non-specific.

Metastasis is the dominant cause of patient death in small-cell lung cancer (SCLC), and a better understanding of the molecular mechanisms underlying SCLC metastasis may potentially improve clinical treatment. Through genome-scale screening for key regulators of mouse Rb1(–/–) Trp53(–/–) SCLC metastasis using the pooled CRISPR/Cas9 library, we identified Cullin5 (CUL5) and suppressor of cytokine signaling 3 (SOCS3), two components of the Cullin-RING E3 ubiquitin ligase complex, as top candidates. Mechanistically, the deficiency of CUL5 or SOCS3 disrupted the functional formation of the E3 ligase complex and prevented the degradation of integrin β1, which stabilized integrin β1 and activated downstream focal adhesion kinase/SRC (FAK/SRC) signaling and eventually drove SCLC metastasis. Low expression levels of CUL5 and SOCS3 were significantly associated with high integrin β1 levels and poor prognosis in a large cohort of 128 clinical patients with SCLC. Moreover, the CUL5-deficient SCLCs were vulnerable to the treatment of the FDA-approved SRC inhibitor dasatinib. Collectively, this work identifies the essential role of CUL5- and SOCS3-mediated integrin β1 turnover in controlling SCLC metastasis, which might have therapeutic implications.

Background: Hepatic stellate cell (HSC) activation induced by transforming growth factor β1 (TGF-β1) plays a pivotal role in fibrogenesis, while the complex downstream mediators of TGF-β1 in such process are largely unknown. Methods: We performed proteomic profiling of mice liver tissues from control, carbon tetrachloride (CCl4)-induced fibrosis and NPLC0393 administrated groups. Our target Mat2a was overexpressed and knocked down in vivo by tail vein injection of AAV vectors. We examined NF-κB transcriptional and MAT2A promoter activation via luciferase assay. Intracellular SAM contents were analyzed by LC/MS method. Findings: We found that methionine adenosyltransferase 2A (MAT2A) is significantly upregulated in the CCl4-induced fibrosis mice model and application of NPLC0393, a known small molecule inhibitor of TGF-β1 signaling pathway, inhibits the upregulation of MAT2A. Mechanistically, TGF-β1 induces phosphorylation of p65 and activation of NF-κB, thereby promoting mRNA transcription and protein expression of MAT2A and reduces S-adenosylmethionine (SAM) concentration in HSCs. Consistently, in vivo and in vitro knockdown of MAT2A alleviates CCl4- and TGF-β1-induced HSC activation, whereas in vivo overexpression of MAT2A facilitates hepatic fibrosis and abolishes therapeutic effect of NPLC0393. Interpretation: This study identifies TGF-β1/p65/MAT2A pathway that is involved in the regulation of intracellular SAM concentration and liver fibrogenesis, suggesting that this pathway is a potential therapeutic target for hepatic fibrosis. Funding: This work was supported by National Natural Science Foundation of China (No. 81500469, 81573873 and 81774196), Zhejiang Provincial Natural Science Foundation of China (No. Y15H030004), the National Key Research and Development Program from the Ministry of Science and Technology of China (No. 2017YFC1700200) and the Key Program of National Natural Science Foundation of China (No. 8153000502). Declaration of Interest: The authors declare no conflict of interest. Ethical Approval: The animal experiment was approved by Science and Technology Commission of Shanghai Municipality, and all experimental procedures were performed according to the ethical guidelines of Animal care and use committee, Shanghai Institute of Materia Medica, Chinese Academy of Sciences.

Increasing evidence has shown that abnormal expression of XPO5 is found in many human cancers and acts as an oncoprotein in certain cancers. However, its functional role in hepatocellular carcinoma (HCC) remains unexplored. In our study, we found that XPO5 was highly expressed in HCC, which was associated with SUMO modification. Moreover, we found that XPO5 was SUMOylated by SUMO2 at K125. Functional experiments revealed that XPO5 SUMOylation could promote MHCC97H cell proliferation, migration and invasion. In addition, we found that the nuclear export of pre-miR-3184 was suppressed by SUMOylated XPO5. Moreover, PLCB1 was identified as the common target of miR-3184-5p and miR-3184-3p. The suppressed phenotype induced by miR-3184-5p and miR-3184-3p could be rescued by overexpression of PLCB1. Bioinformatics analysis showed that PLCB1 expression had a negative relationship with HCC patient survival. The inhibitory effects of MHCC97H cells resulted from abnormal XPO5 SUMO modification could be blocked by miR-3184 inhibitor or PLCB1 overexpression. In conclusion, our findings demonstrate a novel mechanism of XPO5 in HCC, that is, the SUMOylated XPO5 acts as an "oncogenic" role in MHCC97H cells proliferation, migration and invasion by controlling the nuclear-cytoplasm transportation of miR-3184, thus up-regulating PLCB1 expression.

Rationale: Hepatic alveolar echinococcosis (HAE) presents a high pathogenicity and case fatality rate. The main treatment for HAE is surgical resection. Giant lesions in the liver and invasion of the pathogen into the retrohepatic inferior vena cava are usually associated with a poor prognosis when radical resection cannot be performed. Patient concerns: A 56-year-old man who underwent hydatidectomy 7 years prior noted a recurrence of HAE. He was subsidized and admitted to our hospital for the purpose of surgical treatment. Diagnosis: By computed tomography, angiography and three-dimensional (3D) computed tomography reconstruction images, multiple, giant HAE with 75% stenosis was confirmed. Interventions: With the 3D visualization technique, we designed the surgical plan and performed radical resection of the lesions, including the invaded inferior vena cava, and maximized retention of normal liver tissue. The abdominal aorta of an organ donor was used for vascular allograft reconstruction. Outcomes: The patient recovered gradually after the operation. He was followed up for 3 months, and the reconstructed vein patency was good. Lessons: The 3D visualization technique combined with a blood vessel allograft allowed us to expand indications for radical resection of extensive HAE.

We performed the first proteogenomic characterization of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) using paired tumor and adjacent liver tissues from 159 patients. Integrated proteogenomic analyses revealed consistency and discordance among multi-omics, activation status of key signaling pathways, and liver-specific metabolic reprogramming in HBV-related HCC. Proteomic profiling identified three subgroups associated with clinical and molecular attributes including patient survival, tumor thrombus, genetic profile, and the liver-specific proteome. These proteomic subgroups have distinct features in metabolic reprogramming, microenvironment dysregulation, cell proliferation, and potential therapeutics. Two prognostic biomarkers, PYCR2 and ADH1A, related to proteomic subgrouping and involved in HCC metabolic reprogramming, were identified. CTNNB1 and TP53 mutation-associated signaling and metabolic profiles were revealed, among which mutated CTNNB1-associated ALDOA phosphorylation was validated to promote glycolysis and cell proliferation. Our study provides a valuable resource that significantly expands the knowledge of HBV-related HCC and may eventually benefit clinical practice.

PTEN is one of the most frequently mutated tumour suppressors and reduction in PTEN protein stability also plays a role in tumorigenesis. Although several ubiquitin ligases for PTEN have been identified, the deubiquitylase for de-polyubiquitylation and stabilization of PTEN is less defined. Here, we report OTUD3 as a deubiquitylase of PTEN. OTUD3 interacts with, de-polyubiquitylates and stabilizes PTEN. Depletion of OTUD3 leads to the activation of Akt signalling, induction of cellular transformation and cancer metastasis. OTUD3 transgenic mice exhibit higher levels of the PTEN protein and are less prone to tumorigenesis. Reduction of OTUD3 expression, concomitant with decreased PTEN abundance, correlates with human breast cancer progression. Furthermore, we identified loss-of-function OTUD3 mutations in human cancers, which either abolish OTUD3 catalytic activity or attenuate the interaction with PTEN. These findings demonstrate that OTUD3 is an essential regulator of PTEN and that the OTUD3-PTEN signalling axis plays a critical role in tumour suppression.

F-box and WD repeat domain-containing 7 (FBW7) is the substrate recognition component of the Skp1-Cul1-F-box (SCF) ubiquitin ligase complex and functions as a major tumor suppressor by targeting various oncoproteins for degradation. Genomic deletion or mutation of FBW7 has frequently been identified in many human cancers but not in pancreatic ductal adenocarcinoma. Thus it is important to know how the tumor suppressive function of FBW7 is impaired in pancreatic cancer. In this study, we first observed that low FBW7 expression correlated significantly with ERK activation in pancreatic cancer clinical samples, primarily due to KRAS mutations in pancreatic cancer. We further showed that ERK directly interacted with FBW7 and phosphorylated FBW7 at Thr205, which sequentially promoted FBW7 ubiquitination and proteasomal degradation. Furthermore, the phospho-deficient T205A FBW7 mutant is resistant to ERK activation and could significantly suppress pancreatic cancer cell proliferation and tumorigenesis. These results collectively demonstrate how the oncogenic KRAS mutation inhibits the tumor suppressor FBW7, thus revealing an important function of KRAS mutations in promoting pancreatic cancer progression.

The autism spectrum disorders (ASDs) are a collection of human neurological disorders with heterogeneous etiologies. Hyperactivity of E3 ubiquitin (Ub) ligase UBE3A, stemming from 15q11-q13 copy number variations, accounts for 1%-3% of ASD cases worldwide, but the underlying mechanisms remain incompletely characterized. Here we report that the functionality of ALDH1A2, the rate-limiting enzyme of retinoic acid (RA) synthesis, is negatively regulated by UBE3A in a ubiquitylation-dependent manner. Excessive UBE3A dosage was found to impair RA-mediated neuronal homeostatic synaptic plasticity. ASD-like symptoms were recapitulated in mice by overexpressing UBE3A in the prefrontal cortex or by administration of an ALDH1A antagonist, whereas RA supplements significantly alleviated excessive UBE3A dosage-induced ASD-like phenotypes. By identifying reduced RA signaling as an underlying mechanism in ASD phenotypes linked to UBE3A hyperactivities, our findings introduce a new vista of ASD etiology and facilitate a mode of therapeutic development against this increasingly prevalent disease.

The X-linked deubiquitinase, USP9X, is implicated in multiple cancers by targeting various substrates. Increased expression of USP9X is observed in non-small-cell lung cancer (NSCLC) and is correlated with poor prognosis. However, the molecular mechanism for USP9X regulation of tumor cell survival and tumorigenesis in NSCLC is less defined. Methods: In this study, chemical labeling, quantitative proteomic screening was applied to analyze A549 cells with or without USP9X RNA interference. Functional in vitro and in vivo experiments were performed to confirm the oncogenic effects of USP9X in NSCLC and to investigate the underlying mechanisms. Results: The resulting data suggested that dual specificity protein kinase TTK is a potential substrate of USP9X. Further experimental evidences confirmed that USP9X stabilized TTK via direct interaction and efficient deubiquitination of TTK on K48 ubiquitin chain. Moreover, knockdown of USP9X or TTK inhibited cell proliferation, migration and tumorigenesis, and the immunohistochemical analysis of clinical NSCLC samples showed that the protein expression levels of USP9X and TTK were significantly elevated and positively correlated in tumor tissues. Conclusions: In summary, our data demonstrated that the USP9X-TTK axis may play a critical role in NSCLC, and could be considered as a potential therapeutic target.

Polyploidy can lead to aneuploidy and tumorigenesis. Here, we report that the Hippo pathway effector Yap promotes the diploid-polyploid conversion and polyploid cell growth through the Akt-Skp2 axis. Yap strongly induces the acetyltransferase p300-mediated acetylation of the E3 ligase Skp2 via Akt signaling. Acetylated Skp2 is exclusively localized to the cytosol, which causes hyper-accumulation of the cyclin-dependent kinase inhibitor p27, leading to mitotic arrest and subsequently cell polyploidy. In addition, the pro-apoptotic factors FoxO1/3 are overly degraded by acetylated Skp2, resulting in polyploid cell division, genomic instability, and oncogenesis. Importantly, the depletion or inactivation of Akt or Skp2 abrogated Hippo signal deficiency-induced liver tumorigenesis, indicating their epistatic interaction. Thus, we conclude that Hippo-Yap signaling suppresses cell polyploidy and oncogenesis through Skp2.

Background The detrimental effect of prolonged cold ischemia time (PCI) on presensitized transplanted graft is conceivable, but the impact of presensitization status of recipient on PCI-mediated graft injury and inflammation is not well defined. Methods Allogeneic skin grafts from BALB/c donors were transplanted into C57BL/6 recipients for presensitization. Syngeneic or allogeneic heterotopic heart transplantations with PCI were performed using C57BL/6 or BALB/c donors for these recipients through different treatments. Results We revealed that PCI could not affect isograft survival but significantly shortened allograft survival in the presensitized recipients. Depletion of regulatory T cells (Tregs) starting 1 day before and after heart transplantation with anti-CD25 monoclonal antibody remarkably induced intragraft Foxp3 gene expression, worsened architecture damage and subepicardial and intramuscle inflammatory cellular infiltration, and caused a dramatic fall of intragraft CD4+/CD8+ ratio, whereas adoptive transfer of exogenous wild-type Tregs or endogenous Tregs promoted by rapamycin had a beneficial effect on preventing the infiltration of T lymphocytes and Gr-1+ neutrophils and reversed intragraft CD4+/CD8+ ratio, preserving cardiac graft architecture. However, their distinct protective mechanisms showed that rapamycin treatment mainly diminished CD4+ T-cell infiltration. Nevertheless, CD4+ still outnumbered CD8+ T cells in the graft, whereas adoptive transfer of Tregs expanded both CD4+ and CD8+ T cells, particularly CD8+ T cells. Conclusion Allogeneic immunoresponses synergistically enhanced PCI effect under presensitized condition. PCI could affect subsequent immunoresponses. Tregs were closely involved in this pathophysiologic process. Our data may pave the way to use Tregs as a novel therapeutic approach to prevent PCI-mediated injury in the presensitized transplant recipients.

Hedgehog signaling plays very important roles in development and cancers. Vertebrates have three transcriptional factors, Gli1, Gli2 and Gli3. Among them, Gli3 is a very special transcriptional factor which closely resembles Cubitus interruptus (Ci, in Drosophila) structurally and functionally as a ‘double agent’ for Shh target gene expression. Here we show that Gli3 full-length, but not the truncated form, can be methylated at K436 and K595. This methylation is specifically catalyzed by Set7, a lysine methyltransferase (KMT). Methylation at K436 and K595 respectively increases the stability and DNA binding ability of Gli3, resulting in an enhancement of Shh signaling activation. Furthermore, functional experiments indicate that the Gli3 methylation contributes to the tumor growth and metastasis in non-small cell lung cancer in vitro and in vivo. Therefore, we propose that Set7 mediated methylation is a novel PTM of Gli3, which positively regulates the transactivity of Gli3 and the activation of Shh signaling. DOI: http://dx.doi.org/10.7554/eLife.15690.001, eLife digest Cells in mammals need to be able to communicate with each other to enable them to work together in tissues and organs. A signaling pathway called Hedgehog signaling plays a crucial role in carrying information between cells in developing embryos, but if it is active at other times it can also promote the development of cancers. The Hedgehog signaling pathway regulates the activity of several proteins, including one called Gli3. When the Hedgehog signaling pathway is not active, Gli3 is able to switch off certain genes in the cells. On the other hand, when the signaling pathway is active, Gli3 changes shape so that it is able to activate its target genes instead. It is thought that this shape change is triggered by the addition (or removal) of chemical tags to Gli3. So far, researchers have reported that several different types of chemical tags can modify the activity of Gli3. However, it is not clear whether another type of chemical tag – known as a methyl tag – is involved in regulating Gli3. Fu et al. studied Hedgehog signaling in mice. The experiments show that an enzyme called Set7 can modify Gli3 by adding methyl tags to certain sites in the protein. This modification makes the protein’s structure more stable and helps it to bind to the target genes. Further experiments show that these methyl groups contribute to the progression of lung cancer. Fu et al.’s findings expand our understanding of how chemical tags can alter the cells’ response to Hedgehog signaling activity. Future challenges are to understand exactly how Set7 and Gli3 interact and to develop drugs that can block this interaction, which may have the potential to treat cancer. DOI: http://dx.doi.org/10.7554/eLife.15690.002

Chemotherapy is an important therapeutic approach for cancer treatment. However, drug resistance is an obstacle that often impairs the successful use of chemotherapies. Therefore, overcoming drug resistance would lead to better therapeutic outcomes for cancer patients. Recently, studies by our own and other groups have demonstrated that there is an intimate correlation between the loss of the F-box and WD repeat domain-containing 7 (FBW7) tumor suppressor and the incurring drug resistance. While loss of FBW7 sensitizes cancer cells to certain drugs, FBW7-/- cells are more resistant to other types of chemotherapies. FBW7 exerts its tumor suppressor function by promoting the degradation of various oncoproteins that regulate many cellular processes, including cell cycle progression, cellular metabolism, differentiation, and apoptosis. Since loss of the FBW7 tumor suppressor is linked to drug resistance, FBW7 may represent a novel therapeutic target to increase drug sensitivity of cancer cells to conventional chemotherapeutics. This paper thus focuses on the new functional aspects of FBW7 in drug resistance.

Interferon (IFN) regulatory factors (IRFs) are a family of transcription mediators involved in the regulation of type I IFN (IFN-alpha/beta) transcription. Among the nine already identified IRFs, IRF-3 is a constitutively and ubiquitously expressed and plays a critical role in the transcriptional activation of type I IFN and IFN-stimulated genes including IFN-alpha1, IFN-beta and RANTES. In the present study, we report the identification of a novel alternatively spliced transcript of murine Irf-3 gene (mIrf-3) designated mIRF-3a. mIRF-3a is ubiquitously present in mouse tissues along with mIRF-3 and could be translocated into the nucleus in uninfected L929 cells. EMSA and reporter assay demonstrated that mIRF-3a binds to ISRE sequences in murine Ifn-beta promoter and represses Ifn-beta promoter activation induced by Newcastle disease virus infection. These results suggest that mIRF-3a may act as a modulator of mIRF-3 functions in vivo.

Elevated Skp2 expression is frequently observed in many tumors including breast and prostate carcinomas. However, the molecular mechanisms underlying elevated Skp2 expression in prostate cancers have not been fully explored. Hyperactivation of the Akt pathway is considered a hallmark of many cancers and it has been reported that activation of the PI3K/Akt pathway enhances p27 destruction. Thus, we hypothesize that sustained Akt activity in prostate cancer cells leads to elevated phosphorylation of Skp2, and subsequently influences Skp2 stability and its oncogenic functions. In support of our hypothesis, we found that Skp2 abundance is affected by manipulation of the PTEN/PI3K/Akt pathway in the PC3 and LNCaP prostate cancer cell lines. Furthermore, we showed that this is partially through phosphorylation of Skp2 by Akt, which impairs Skp2 destruction by Cdh1. Sequence analysis revealed that the Akt phosphorylation site (Ser72) is localized in a putative Nuclear Localization Sequence (NLS). Consistent with this notion, we found that overexpression of Myr-Akt promoted Skp2 cytoplasmic translocation, and that inhibition of PI3K/Akt activity enhanced its nuclear localization. We believe that these studies will provide a novel mechanism for Skp2 overexpression in prostate cancers and provide the rationale for developing Akt1-specific inhibitors to treat prostate cancer patients.

Breast cancer is the most common type of cancer among American women, and remains the second leading cause of cancer-related death for female in the United States. It has been known that several signaling pathways and various factors play critical roles in the development and progression of breast cancer, such as estrogen receptor, Notch, PTEN, human epidermal growth factor receptor 2, PI3K/Akt, BRCA1, and BRCA2. Emerging evidence has shown that the F-box protein S-phase kinase associated protein 2 (Skp2) also plays an important role in the pathogenesis of breast cancer. Therefore, in this brief review, we summarize the novel functions of Skp2 in the pathogenesis of breast cancer. Moreover, we provide further evidence regarding the state of our knowledge toward the development of novel Skp2 inhibitors especially natural "chemopreventive agents" as targeted approach for the prevention and/or treatment of breast cancer.

For raising the antigenicity of Mycobacterium bovis single antigen, fusion protein of two genes was acquired. The DNA fragments of ag85b and mpb64 were fused by splicing by overlapping extension (SOE) polymerase chain reaction(PCR), and the fusion gene ag85b-mpb64 were cloned into pMD-18-T vector, then we got the recombinant plasmid pMD-85b-64. pMD-85b-64 and pET28a(+) were digested by BamH I and EcoR I double enzymes. The purified pMD-85b-64 fusion gene was subcloned into the expression vector pET28a(+), and the prokaryotic expression vector pET-85b-64 was constructed. Plasmid containing pET-85b-64 was transformed into competence Escherichia coli BL21 (DE3). The bacterium was induced by isopropyl-β-D-thiogalactopyranoside(IPTG) and its lysates were loaded directly onto sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), approximately 58 ku exogenous protein was observed on the SDS-PAGE. The protein was analyzed by using Western blotting, the results indicated that the protein was of antigenic activity of Mycobacterium bovis. These results could serve as a basis for further studies on the usefulness of the fusion gene and its expression product in the development of subunit vaccine and DNA vaccine against bovine tuberculosis.