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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

The deubiquitinase USP9X is involved in multiple diseases including neurodegeneration, epilepsy, and various types of tumors by targeting different substrates. In the present study, we aimed to explore the potential substrates of USP9X and performed SILAC-based quantitative proteomics to compare these substrates in USP9X-knockdown and wild-type HeLa cells. We consequently carried out Flag-NFX1-123 tag affinity-based mass spectrometry and confirmed that the X-box binding nuclear factor NFX1-123 interacted with USP9X. Moreover, immunoprecipitation assays verified a direct interaction between USP9X and NFX1-123. Further experiments confirmed that NFX1-123 could be modified by ubiquitination and that USP9X stabilized NFX1-123 via efficient deubiquitination of NFX1-123. Knockdown of USP9X resulted in decreased NFX1-123 protein levels compared with their unchanged corresponding mRNA levels in different cell lines. In summary, we found that NFX1-123 was a bona fide substrate of the deubiquitinase USP9X and that it could be degraded by the ubiquitin-proteasome system. The present study provided new insight into understanding the biological function of USP9X by targeting its substrate NFX1-123.

Oligosaccharides have important therapeutic applications. A useful route for oligosaccharides synthesis is reverse hydrolysis by β-glucosidase. However, the low conversion efficiency of disaccharides from monosaccharides limits its large-scale production because the equilibrium is biased in the direction of hydrolysis. Based on the analysis of the docking results, we hypothesized that the hydropathy index of key amino acid residues in the catalytic site is closely related with disaccharide synthesis and more hydrophilic residues located in the catalytic site would enhance reverse hydrolysis activity. In this study, positive variants TrCel1bI177S, TrCel1bI177S/I174S, and TrCel1bI177S/I174S/W173H, and one negative variant TrCel1bN240I were designed according to the Hydropathy Index For Enzyme Activity (HIFEA) strategy. The reverse hydrolysis with TrCel1bI177S/I174S/W173H was accelerated and then the maximum total production (195.8 mg/mL/mg enzyme) of the synthesized disaccharides was increased by 3.5-fold compared to that of wildtype. On the contrary, TrCel1bN240I lost reverse hydrolysis activity. The results demonstrate that the average hydropathy index of the key amino acid residues in the catalytic site of TrCel1b is an important factor for the synthesis of laminaribiose, sophorose, and cellobiose. The HIFEA strategy provides a new perspective for the rational design of β-glucosidases used for the synthesis of oligosaccharides.

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.

Alterations in cellular ubiquitin (Ub) homeostasis, known as Ub stress, feature and affect cellular responses in multiple conditions, yet the underlying mechanisms are incompletely understood. Here we report that autophagy receptor p62/sequestosome-1 interacts with E2 Ub conjugating enzymes, UBE2D2 and UBE2D3. Endogenous p62 undergoes E2-dependent ubiquitylation during upregulation of Ub homeostasis, a condition termed as Ub+ stress, that is intrinsic to Ub overexpression, heat shock or prolonged proteasomal inhibition by bortezomib, a chemotherapeutic drug. Ubiquitylation of p62 disrupts dimerization of the UBA domain of p62, liberating its ability to recognize polyubiquitylated cargoes for selective autophagy. We further demonstrate that this mechanism might be critical for autophagy activation upon Ub+ stress conditions. Delineation of the mechanism and regulatory roles of p62 in sensing Ub stress and controlling selective autophagy could help to understand and modulate cellular responses to a variety of endogenous and environmental challenges, potentially opening a new avenue for the development of therapeutic strategies against autophagy-related maladies.

Lin28a, originally discovered in the nematode Caenorhabditis elegans and highly conserved across species, is a well characterized regulator of let-7 microRNA (miRNA) and is implicated in cell proliferation and pluripotency control. However, little is known about how Lin28a function is modulated at the post-translational level and thereby responds to major signaling pathways. Here we show that Lin28a is directly phosphorylated by ERK1/2 kinases at Ser-200. By editing lin28a gene with the CRISPR/Cas9-based method, we generated P19 mouse embryonic carcinoma stem cells expressing Lin28a-S200A (phospho-deficient) and Lin28a-S200D (phospho-mimetic) mutants, respectively, to study the functional impact of Ser-200 phosphorylation. Lin28a-S200D-expressing cells, but not Lin28a-S200A-expressing or control P19 embryonic carcinoma cells, displayed impaired inhibition of let-7 miRNA and resulted in decreased cyclin D1, whereas Lin28a-S200A knock-in cells expressed less let-7 miRNA, proliferated faster, and exhibited differentiation defect upon retinoic acid induction. Therefore our results support that ERK kinase-mediated Lin28a phosphorylation may be an important mechanism for pluripotent cells to facilitate the escape from the self-renewal cycle and start the differentiation process.

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).

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.

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.

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.

Growth signals, such as extracellular nutrients and growth factors, have substantial effects on genome integrity; however, the direct underlying link remains unclear. Here, we show that the mechanistic target of rapamycin (mTOR)-ribosomal S6 kinase (S6K) pathway, a central regulator of growth signalling, phosphorylates RNF168 at Ser60 to inhibit its E3 ligase activity, accelerate its proteolysis and impair its function in the DNA damage response, leading to accumulated unrepaired DNA and genome instability. Moreover, loss of the tumour suppressor liver kinase B1 (LKB1; also known as STK11) hyperactivates mTOR complex 1 (mTORC1)-S6K signalling and decreases RNF168 expression, resulting in defects in the DNA damage response. Expression of a phospho-deficient RNF168-S60A mutant rescues the DNA damage repair defects and suppresses tumorigenesis caused by Lkb1 loss. These results reveal an important function of mTORC1-S6K signalling in the DNA damage response and suggest a general mechanism that connects cell growth signalling to genome stability control.

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.

Abnormal activation of the oncogenic E3 ubiquitin ligase murine double minute 2 (MDM2) is frequently observed in human cancers. By ubiquitinating the tumor suppressor p53 protein, which leads to its proteasome-mediated destruction, MDM2 limits the tumor-suppressing activity of p53. On the other hand, by ubiquitinating itself, MDM2 targets itself for destruction and promotes the p53 tumor suppressor pathway, a process that can be antagonized by the deubiquitinase herpesvirus-associated ubiquitin-specific protease (HAUSP). We investigated the regulation of MDM2 substrate specificity and found that acetyltransferase p300–mediated acetylation and stabilization of MDM2 are molecular switches that block self-ubiquitination, thereby shifting its E3 ligase activity toward p53. In vitro and in cancer cell lines, p300-mediated acetylation of MDM2 on Lys182 and Lys185 enabled HAUSP to bind, presumably deubiquitinate, and stabilize MDM2. This acetylation within the nuclear localization signal domain decreased its interaction with the acidic domain, subsequently increased the interaction between the acidic domain and RING domain in MDM2, enabled the binding of HAUSP to the acidic domain in MDM2, and shifted MDM2 activity from autoubiquitination to p53 ubiquitination. However, upon genotoxic stress through exposure to etoposide, the deacetylase sirtuin 1 (SIRT1) deacetylated MDM2 at Lys182 and Lys185, thereby promoting self-ubiquitination and less ubiquitination and subsequent degradation of p53, thus increasing p53-dependent apoptosis. Therefore, this study indicates that dynamic acetylation is a molecular switch in the regulation of MDM2 substrate specificity, revealing further insight into the posttranslational regulation of the MDM2/p53 cell survival axis.

The centromere regulates proper chromosome segregation, and its dysfunction is implicated in chromosomal instability (CIN). However, relatively little is known about how centromere dysfunction occurs in cancer. Here, we define the consequences of phosphorylation by cyclin E1/CDK2 on a conserved Ser18 residue of centromere-associated protein CENP-A, an essential histone H3 variant that specifies centromere identity. Ser18 hyperphosphorylation in cells occurred upon loss of FBW7, a tumor suppressor whose inactivation leads to CIN. This event on CENP-A reduced its centromeric localization, increased CIN, and promoted anchorage-independent growth and xenograft tumor formation. Overall, our results revealed a pathway that cyclin E1/CDK2 activation coupled with FBW7 loss promotes CIN and tumor progression via CENP-A–mediated centromere dysfunction. Cancer Res; 77(18); 4881–93. ©2017 AACR.

Wei and colleagues report that phosphorylation of Sin1 by S6K or Akt results in its dissociation from mTORC2, thus suppressing mTORC2 activity. A cancer-patient-derived Sin1 mutation that impairs this phosphorylation leads to mTORC2 hyperactivation and increased tumour formation in mice.

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

The E3 ubiquitin ligase β-TRCP, acting in concert with casein kinase I, drives ubiquitination and degradation of VEGFR2, and renders human papillary thyroid cancer cells resistant to the VEGFR2 inhibitor sorafenib., The incidence of human papillary thyroid cancer (PTC) is increasing and an aggressive subtype of this disease is resistant to treatment with vascular endothelial growth factor receptor 2 (VEGFR2) inhibitor. VEGFR2 promotes angiogenesis by triggering endothelial cell proliferation and migration. However, the molecular mechanisms governing VEGFR2 stability in vivo remain unknown. Additionally, whether VEGFR2 influences PTC cell migration is not clear. We show that the ubiquitin E3 ligase SCFβ-TRCP promotes ubiquitination and destruction of VEGFR2 in a casein kinase I (CKI)–dependent manner. β-TRCP knockdown or CKI inhibition causes accumulation of VEGFR2, resulting in increased activity of signaling pathways downstream of VEGFR2. β-TRCP–depleted endothelial cells exhibit enhanced migration and angiogenesis in vitro. Furthermore, β-TRCP knockdown increased angiogenesis and vessel branching in zebrafish. Importantly, we found an inverse correlation between β-TRCP protein levels and angiogenesis in PTC. We also show that β-TRCP inhibits cell migration and decreases sensitivity to the VEGFR2 inhibitor sorafenib in poorly differentiated PTC cells. These results provide a new biomarker that may aid a rational use of tyrosine kinase inhibitors to treat refractory PTC.

Deregulation of the mammalian target of rapamycin (mTOR) signaling pathway has been found in a variety of human cancers. However, the exact molecular mechanism how the mTOR signaling pathway is regulated remains largely elusive. Recently, DEPTOR was identified as an endogenous mTOR inhibitor that could suppress mTOR activity in vivo. More importantly, accumulated evidence has implicated that DEPTOR plays a pivotal role in the development and progression of human malignances, which could in part be mediated through its inhibitory role toward mTOR. Furthermore, three independent laboratories including our own have demonstrated that the stability of DEPTOR is controlled by the SCFβ-TrCP E3 ubiquitin ligase and deregulated DEPTOR destruction might contribute to hyperactivation of mTOR in pathologic conditions including cancer. This review discusses the recent literature regarding the function of DEPTOR involved in cell growth, apoptosis, autophagy, epithelial-mesenchymal transition, and drug resistance, all of which are associated with the pathogenesis of human cancers. Moreover, we also summarize that targeting DEPTOR may be a novel strategy for achieving better anticancer treatments.

SummaryThe NFκB/Rel family of proteins play critical roles in a variety of cellular processes. Thus, their physiological activation is tightly controlled. Recently, the NFκB2/p100 precursor has been characterized as the fourth IκB type of suppressor for NFκB. However, the molecular mechanism(s) underlying regulated destruction of NFκB2 remains largely unknown. Here, we report that, unlike other IκBs, ubiquitination and destruction of NFκB2 are governed by SCFFbw7 in a GSK3-dependent manner. In Fbw7−/− cells, elevated expression of NFκB2/p100 leads to a subsequent reduction in NFκB signaling pathways and elevated sensitivity to TNFα-induced cell death. Reintroducing wild-type Fbw7, but not disease-derived mutant forms of Fbw7, rescues NFκB activity. Furthermore, T cell-specific depletion of Fbw7 also leads to reduced NFκB activity and perturbed T cell differentiation. Therefore, our work identifies Fbw7 as a physiological E3 ligase controlling NFκB2′s stability. It further implicates that Fbw7 might exert its tumor-suppressor function by regulating NFκB activity.

This article describes a functional interaction between PP2A and FOXO3a in which PP2A promotes rapid dephosphorylation of FOXO3a at its conserved AKT phosphorylation sites, leading to FOXO3a dissociation from 14-3-3, nuclear translocation, and transcriptional activation in response to inhibition of PI3K signaling., Forkhead box transcription factor FOXO3a, a key regulator of cell survival, is regulated by reversible phosphorylation and subcellular localization. Although the kinases regulating FOXO3a activity have been characterized, the role of protein phosphatases (PP) in the control of FOXO3a subcellular localization and function is unknown. In this study, we detected a robust interaction between FOXO3a and PP2A. We further demonstrate that 14-3-3, while not impeding the interaction between PP2A and FOXO3a, restrains its activity toward AKT phosphorylation sites T32/S253. Disruption of PP2A function revealed that after AKT inhibition, PP2A-mediated dephosphorylation of T32/S253 is required for dissociation of 14-3-3, nuclear translocation, and transcriptional activation of FOXO3a. Our findings reveal that distinct phosphatases dephosphorylate conserved AKT motifs within the FOXO family and that PP2A is entwined in a dynamic interplay with AKT and 14-3-3 to directly regulate FOXO3a subcellular localization and transcriptional activation.

The Set8/PR-Set7/KMT5a methyltransferase plays critical roles in governing transcriptional regulation, cell cycle progression and tumorigenesis. Although CRL4Cdt2 was reported to regulate Set8 stability, deleting the PIP motif only led to partial resistance to ultraviolet-induced degradation of Set8, indicating the existence of additional E3 ligase(s) controlling Set8 stability. Furthermore, it remains largely undefined how DNA damage-induced kinase cascades trigger the timely destruction of Set8 to govern tumorigenesis. Here, we report that SCFβ-TRCP earmarks Set8 for ubiquitination and degradation in a casein kinase I-dependent manner, which is activated by DNA-damaging agents. Biologically, both CRL4Cdt2 and SCFβ-TRCP-mediated pathways contribute to ultraviolet-induced Set8 degradation to control cell cycle progression, governing the onset of DNA damage-induced checkpoints. Therefore, like many critical cell cycle regulators including p21 and Cdt1, we uncover a tight regulatory network to accurately control Set8 abundance. Our studies further suggest that aberrancies in this delicate degradation pathway might contribute to aberrant elevation of Set8 in human tumours., The Set8-Set7 methyltransferase plays a critical role in cell cycle progression and tumorigenesis, and is degraded through modification by the E3 ubiquitin ligase CRL4Cdt2. Here Wang et al. identify SCFβ-TRCP as an additional E3 ubiquitin ligase that marks Set8 for degradation in response to DNA damage.

Summary Mdm2 is the major negative regulator of the p53 pathway. Here, we report that Mdm2 is rapidly degraded after DNA damage and that phosphorylation of Mdm2 by casein kinase I (CKI) at multiple sites triggers its interaction with, and subsequent ubiquitination and destruction, by SCF β-TRCP . Inactivation of either β-TRCP or CKI results in accumulation of Mdm2 and decreased p53 activity, and resistance to apoptosis induced by DNA damaging agents. Moreover, SCF β-TRCP -dependent Mdm2 turnover also contributes to the control of repeated p53 pulses in response to persistent DNA damage. Our results provide insight into the signaling pathways controlling Mdm2 destruction and further suggest that compromised regulation of Mdm2 results in attenuated p53 activity, thereby facilitating tumor progression.