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The VHL protein (pVHL) functions as a tumor suppressor by regulating the degradation or activation of protein substrates such as HIF1α and Akt. In human cancers harboring wild-type VHL, the aberrant downregulation of pVHL is frequently detected and critically contributes to tumor progression. However, the underlying mechanism by which the stability of pVHL is deregulated in these cancers remains elusive. Here, we identify cyclin-dependent kinase 1 (CDK1) and peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1) as two previously uncharacterized regulators of pVHL in multiple types of human cancers harboring wild-type VHL including triple-negative breast cancer (TNBC). PIN1 and CDK1 cooperatively modulate the protein turnover of pVHL, thereby conferring tumor growth, chemotherapeutic resistance and metastasis both in vitro and in vivo. Mechanistically, CDK1 directly phosphorylates pVHL at Ser80, which primes the recognition of pVHL by PIN1. PIN1 then binds to phosphorylated pVHL and facilitates the recruitment of the E3 ligase WSB1, therefore targeting pVHL for ubiquitination and degradation. Furthermore, the genetic ablation or pharmacological inhibition of CDK1 by RO-3306 and PIN1 by all-trans retinoic acid (ATRA), the standard care for Acute Promyelocytic Leukemia could markedly suppress tumor growth, metastasis and sensitize cancer cells to chemotherapeutic drugs in a pVHL dependent manner. The histological analyses show that PIN1 and CDK1 are highly expressed in TNBC samples, which negatively correlate with the expression of pVHL. Taken together, our findings reveal the previous unrecognized tumor-promoting function of CDK1/PIN1 axis through destabilizing pVHL and provide the preclinical evidence that targeting CDK1/PIN1 is an appealing strategy in the treatment of multiple cancers with wild-type VHL.

ObjectiveHaematogenous dissemination is a prevalent route of colorectal cancer (CRC) metastasis. However, as the gatekeeper of vessels, the role of tumour pericytes (TPCs) in haematogenous metastasis remains largely unknown. Here, we aimed to investigate the heterogeneity of TPCs and their effects on CRC metastasis.DesignTPCs were isolated from patients with CRC with or without liver metastases and analysed by single-cell RNA sequencing (scRNA-seq). Clinical CRC specimens were collected to analyse the association between the molecular profiling of TPCs and CRC metastasis. RNA-sequencing, chromatin immunoprecipitation-sequencing and bisulfite-sequencing were performed to investigate the TCF21-regulated genes and mechanisms underlying integrin α5 onTCF21DNA hypermethylation. Pericyte-conditionalTcf21-knockout mice were constructed to investigate the effects of TCF21 in TPCs on CRC metastasis. Masson staining, atomic force microscopy, second-harmonic generation and two-photon fluorescence microscopy were employed to observe perivascular extracellular matrix (ECM) remodelling.ResultsThirteen TPC subpopulations were identified by scRNA-seq. A novel subset of TCF21highTPCs, termed ‘matrix–pericytes’, was associated with liver metastasis in patients with CRC. TCF21 in TPCs increased perivascular ECM stiffness, collagen rearrangement and basement membrane degradation, establishing a perivascular metastatic microenvironment to instigate colorectal cancer liver metastasis (CRCLM).Tcf21depletion in TPCs mitigated perivascular ECM remodelling and CRCLM, whereas the coinjection of TCF21highTPCs and CRC cells markedly promoted CRCLM. Mechanistically, loss of integrin α5 inhibited the FAK/PI3K/AKT/DNMT1 axis to impairTCF21DNA hypermethylation in TCF21highTPCs.ConclusionThis study uncovers a previously unidentified role of TPCs in haematogenous metastasis and provides a potential diagnostic marker and therapeutic target for CRC metastasis.

Supplemental data includes supplement materials, methods and data

Supplemental Fig.S1. HEATR1 regulates cancer cell response to chemotherapy

Elucidating mechanisms of chemoresistance is critical to improve cancer therapy, especially for the treatment of pancreatic ductal adenocarcinoma (PDAC). Genome-wide association studies have suggested the less studied gene HEAT repeat-containing protein 1 (HEATR1) as a possible determinant of cellular sensitivity to different chemotherapeutic drugs. In this study, we assessed this hypothesized link in PDAC, where HEATR1 expression is downregulated significantly. HEATR1 silencing in PDAC cells increased resistance to gemcitabine and other chemotherapeutics, where this effect was associated with increased AKT kinase phosphorylation at the Thr308 regulatory site. Mechanistically, HEATR1 enhanced cell responsiveness to gemcitabine by acting as a scaffold to facilitate interactions between AKT and the protein phosphatase PP2A, thereby promoting Thr308 dephosphorylation. Consistent with these findings, treatment with the AKT inhibitor triciribine sensitized HEATR1-depleted PDAC cells to gemcitabine, suggesting that this therapeutic combination may overcome gemcitabine resistance in patients with low HEATR1 expression. Clinically, we found that HEATR1 downregulation in PDAC patients was associated with increased AKT phosphorylation, poor response to tumor resection plus gemcitabine standard-of-care treatment, and shorter overall survival. Collectively, our findings establish HEATR1 as a novel regulator of AKT and a candidate predictive and prognostic indicator of drug responsiveness and outcome in PDAC patients. Cancer Res; 76(3); 572–81. ©2015 AACR.

Hypoxia-inducible factor 2α (HIF2α) plays a pivotal role in breast tumor growth and metastasis. However, the regulatory mechanisms of HIF2α protein stability remain poorly understood. The precise role of the deubiquitinase (DUB) ubiquitin-specific peptidase 5 (USP5) in breast cancer and the underlying mechanism remains largely unknown. Here, we identified USP5 as a novel DUB for HIF2α. Physically, USP5 interacts with HIF2α and protects HIF2α from ubiquitin-proteasome degradation, thereby promoting the transcription of HIF2α target genes, such as SLC2A1, PLOD2, P4HA1, and VEGFA. USP5 ablation impairs breast cancer cells proliferation, colony formation, migration, and invasion. Moreover, USP5 is highly expressed in breast cancer, and the protein levels of USP5 are positively correlated with HIF2α protein levels in human breast cancer tissues. Importantly, high levels of USP5 leads to poor clinical outcome in patients with breast cancer. Collectively, USP5 stabilizes HIF2α through its DUB activity and provides a potential therapeutic target for breast cancer.

Aberrant angiogenesis is a hallmark of cancer and is critically associated with tumor progression. Perivascular cells are essential components of blood vessels, and the role of tumor perivascular cell-derived extracellular vesicles (TPC-EVs) in angiogenesis remains elusive. In the present study, using genetic mouse models and pharmacological inhibitors, we found that ablation of perivascular cells inhibited angiogenesis in allografted colorectal cancer tumors. Further studies demonstrated that TPC-EVs promoted the proliferation, migration, invasion, viability, and tube formation of HUVECs. They also facilitated vessel spouting in rat aortic rings and induced neovascularization in chick chorioallantoic membranes (CAMs). Silencing of Gas6 or blockade of the Axl pathway suppressed TPC-EV-induced angiogenesis in vitro and ex vivo. Moreover, inhibition of the Gas6/Axl signaling pathway impaired TPC-EV-mediated angiogenesis in vivo. Our findings present a deeper insight into the biological functions of TPCs and TPC-EVs in tumor angiogenesis and demonstrate that TPC-EV-derived Gas6 could be an attractive and innovative regulator of tumor angiogenesis.

The relationship between chronic psychological stress and tumorigenesis has been well defined in epidemiological studies; however, the underlying mechanism remains underexplored. In this study, we discovered that impaired macrophage phagocytosis contributed to the psychological stress-evoked tumor susceptibility, and the stress hormone glucocorticoid (GC) was identified as a principal detrimental factor. Mechanistically, GC disturbed the balance of the “eat me” signal receptor (low-density lipoprotein receptor-related protein-1, LRP1) and the “don't eat me” signal receptor (signal regulatory protein alpha, SIRPα). Further analysis revealed that GC led to a direct, glucocorticoid receptor (GR)-dependent trans-repression of LRP1 expression, and the repressed LRP1, in turn, resulted in the elevated gene level of SIRPα by down-regulating miRNA-4695-3p. These data collectively demonstrate that stress induces the imbalance of the LRP1/SIRPα axis and entails the disturbance of tumor cell clearance by macrophages. Our findings provide the mechanistic insight into psychological stress-evoked tumor susceptibility and indicate that the balance of LRP1/SIRPα axis may serve as a potential therapeutic strategy for tumor treatment.

Breast cancer is one of the most common malignancies in women worldwide. Triple-negative breast cancer (TNBC) is a highly aggressive and metastatic subtype that has the characteristics of easy recurrence, poor prognosis as well as lack of targeted therapeutics. Snail1, a key factor regulating epithelial-mesenchymal transition (EMT) process, contributing to metastasis and chemoresistance in human cancers. However, the molecular mechanism of Snail1 stabilization in cancers is not fully understood. Here, we demonstrate that the deubiquitinating enzyme USP9X deubiquitinates and stabilizes Snail1, thereby promoting metastasis and chemoresistance. The depletion and pharmacological inhibition of USP9X by WP1130, an inhibitor of USP9X, downregulate endogenous Snail1 protein, inhibit cell migration, invasion, metastasis, and increase cellular sensitivity to cisplatin and paclitaxel both in vitro and in vivo, whereas the reconstitution of Snail1 in cells with USP9X depletion at least partially reverses these phenotypes. Overall, our study establishes the USP9X-Snail1 axis as an important regulatory mechanism of breast cancer metastasis and chemoresistance and provides a rationale for potential therapeutic interventions in the treatment of TNBC.

Vessel co-option has been demonstrated to mediate colorectal cancer liver metastasis (CRCLM) resistance to antiangiogenic therapy. The current mechanisms underlying vessel co-option have mainly focused on "hijacker" tumor cells, whereas the function of the "hijackee" sinusoidal blood vessels has not been explored. Here, we found that the occurrence of vessel co-option in bevacizumab-resistant CRCLM xenografts was associated with increased expression of fibroblast activation protein α (FAPα) in the co-opted hepatic stellate cells (HSCs), which was dramatically attenuated in HSC-specific conditional Fap-knockout mice bearing CRCLM allografts. Mechanistically, bevacizumab treatment induced hypoxia to upregulate the expression of fibroblast growth factor-binding protein 1 (FGFBP1) in tumor cells. Gain- or loss-of-function experiments revealed that the bevacizumab-resistant tumor cell-derived FGFBP1 induced FAPα expression by enhancing the paracrine FGF2/FGFR1/ERK1/-2/EGR1 signaling pathway in HSCs. FAPα promoted CXCL5 secretion in HSCs, which activated CXCR2 to promote the epithelial-mesenchymal transition of tumor cells and the recruitment of myeloid-derived suppressor cells. These findings were further validated in tumor tissues derived from patients with CRCLM. Targeting FAPα+ HSCs effectively disrupted the co-opted sinusoidal blood vessels and overcame bevacizumab resistance. Our study highlights the role of FAPα+ HSCs in vessel co-option and provides an effective strategy to overcome the vessel co-option-mediated bevacizumab resistance.

Exosomes are attractive as nucleic-acid carriers because of their favourable pharmacokinetic and immunological properties and their ability to penetrate physiological barriers that are impermeable to synthetic drug-delivery vehicles. However, inserting exogenous nucleic acids, especially large messenger RNAs, into cell-secreted exosomes leads to low yields. Here we report a cellular-nanoporation method for the production of large quantities of exosomes containing therapeutic mRNAs and targeting peptides. We transfected various source cells with plasmid DNAs and stimulated the cells with a focal and transient electrical stimulus that promotes the release of exosomes carrying transcribed mRNAs and targeting peptides. Compared with bulk electroporation and other exosome-production strategies, cellular nanoporation produced up to 50-fold more exosomes and a more than 103-fold increase in exosomal mRNA transcripts, even from cells with low basal levels of exosome secretion. In orthotopic phosphatase and tensin homologue (PTEN)-deficient glioma mouse models, mRNA-containing exosomes restored tumour-suppressor function, enhanced inhibition of tumour growth and increased survival. Cellular nanoporation may enable the use of exosomes as a universal nucleic-acid carrier for applications requiring transcriptional manipulation.

Background: N6-methyladenosine (m6A) is related to the progression of multiple cancers. However, the underlying influences of m6A-associated genes on the tumor immune microenvironment in hepatocellular carcinoma (HCC) remain poorly understood. Therefore, we sought to construct a survival prediction model using m6A-associated genes to clarify the molecular and immune characteristics of HCC.Methods: HCC case data were downloaded from The Cancer Genome Atlas (TCGA). Then, by applying consensus clustering, we identified two distinct HCC clusters. Next, four m6A-related genes were identified to construct a prognostic model, which we validated with Gene Expression Omnibus (GEO) and International Cancer Genome Consortium (ICGC) datasets. Additionally, the molecular and immune characteristics in different subgroups were analyzed.Results: m6A RNA methylation regulators were differentially expressed between HCC and normal samples and linked with immune checkpoint expression. Using consensus clustering, we divided HCC samples into two subtypes with distinct clinical features. Cluster 2 was associated with unfavorable prognosis, higher immune checkpoint expression and immune cell infiltration levels. In addition, the immune and carcinogenic signaling pathways were enriched in cluster 2. Furthermore, we constructed a risk model using four m6A-associated genes. Patients with different risk scores had distinct survival times, expression levels of immunotherapy biomarkers, TP53 mutation rates, and sensitivities to chemotherapy and targeted therapy. Similarly, the model exhibited an identical impact on overall survival in the validation cohorts.Conclusion: The constructed m6A-based signature may be promising as a biomarker for prognostics and to distinguish immune characteristics in HCC.

Sepsis is a major cause of patient mortality and morbidity from bacterial infections. Although neutrophils are known to be important in the development of sepsis, how distinctive neutrophil subtypes regulate inflammatory processes involved in septicemia remains unclear. Preconditioning protects organisms against subsequent higher-dose exposures to the same, or even different, stimuli. Several studies have reported various effects of preconditioning on immune cells. However, the detailed mechanisms underlying neutrophil-mediated protection through preconditioning in sepsis remain unknown. Methods: Flow cytometry was conducted to sort the mice peritoneal lavage cells and the blood samples from patients with sepsis. Western blotting and ELISA were carried out to elucidate the expression of TLR9 signal transduction pathway proteins. Histological analysis was used to assess the effect of InP on intestine and liver structure in tlr9-/- and cav-1-/- mice. Fluorescence microscopy, Co-IP, and FRET were carried out to determine the association of TLR9 with Cav-1. Results: We show that membrane toll-like receptor-9 positive (mTLR9+) neutrophils exert a protective effect against fatal bacterial infections through the process of inflammatory preconditioning (InP). InP, which occurs in the setting of a low-dose bacterial challenge, active ingredient is Monophosphoryl lipid A (MPLA), triggers the membrane translocation of TLR9 from the neutrophil cytosol, where it binds to Cav-1. Our findings showed that InP enables TLR9 to facilitate MyD88-mediated TRAF3 and IRF3 signal transduction. Depletion of either TLR9 or Cav-1 largely eliminates the neutrophil-mediated InP effect in sepsis models in vitro and in vivo. Further, examination of clinical samples from patients with sepsis showed that clinical outcomes and likelihood of recovery are closely correlated with mTLR9 and Cav-1 expression in circulating neutrophils. Conclusion: These results demonstrate that the TLR9-Cav-1 axis is a critical signaling pathway involved in the regulation of neutrophil-dependent MPLA mediated InP, and the presence of mTLR9+ neutrophils could be an attractive indicator of clinical outcomes in bacterial sepsis that could be further explored as a potential therapeutic target.

The relationship between chronic psychological stress and tumorigenesis has been well defined in epidemiological studies; however, the underlying mechanism remains underexplored. In this study, we discovered that impaired macrophage phagocytosis contributed to the psychological stress-evoked tumor susceptibility, and the stress hormone glucocorticoid (GC) was identified as a principal detrimental factor. Mechanistically, GC disturbed the balance of the “eat me” signal receptor (low-density lipoprotein receptor-related protein-1, LRP1) and the “don't eat me” signal receptor (signal regulatory protein alpha, SIRPα). Further analysis revealed that GC led to a direct, glucocorticoid receptor (GR)-dependent trans-repression of LRP1 expression, and the repressed LRP1, in turn, resulted in the elevated gene level of SIRPα by down-regulating miRNA-4695-3p. These data collectively demonstrate that stress induces the imbalance of the LRP1/SIRPα axis and entails the disturbance of tumor cell clearance by macrophages. Our findings provide the mechanistic insight into psychological stress-evoked tumor susceptibility and indicate that the balance of LRP1/SIRPα axis may serve as a potential therapeutic strategy for tumor treatment., Graphical abstract Stress-induced glucocorticoid inhibits the expression of “eat me” signal receptor LRP1 by GR-dependent trans-repression, which in turn raises the protein level of “don't eat me” signal receptor SIRPα in an miRNA-4695-3p-dependent manner. The imbalance of LRP1 and SIRPα disrupted by glucocorticoid thus suppresses the phagocytosis of tumor cells by macrophages.Image 1

Antiangiogenic tyrosine kinase inhibitors (AA‐TKIs) have become a promising therapeutic strategy for colorectal cancer (CRC). In clinical practice, a significant proportion of cancer patients temporarily discontinue AA‐TKI treatment due to recurrent toxicities, economic burden or acquired resistance. However, AA‐TKI therapy withdrawal‐induced tumour revascularization frequently occurs, hampering the clinical application of AA‐TKIs. Here, this study demonstrates that tumour perivascular cells mediate tumour revascularization after withdrawal of AA‐TKI therapy. Pharmacological inhibition and genetic ablation of perivascular cells largely attenuate the rebound effect of CRC vascularization in the AA‐TKI cessation experimental settings. Mechanistically, tumour perivascular cell‐derived extracellular vehicles (TPC‐EVs) contain Gas6 that instigates the recruitment of endothelial progenitor cells (EPCs) for tumour revascularization via activating the Axl pathway. Gas6 silence and an Axl inhibitor markedly inhibit tumour revascularization by impairing EPC recruitment. Consequently, combination therapy of regorafenib with the Axl inhibitor improves overall survival in mice metastatic CRC model by inhibiting tumour growth. Together, these data shed new mechanistic insights into perivascular cells in off‐AA‐TKI‐induced tumour revascularization and indicate that blocking the Axl signalling may provide an attractive anticancer approach for sustaining long‐lasting angiostatic effects to improve the therapeutic outcomes of antiangiogenic drugs in CRC.

KRAS, the most frequently mutated oncogene, plays a predominant role in driving initiation and progression of cancers. Decades of effort to target KRAS using small molecules has been unsuccessful, causing KRAS to be considered an "undruggable" cancer target. However, this view began to change recently, as drug discovery techniques have developed several KRAS G12C allosteric inhibitors that are currently being evaluated in clinical trials. Herein we provide an in-depth analysis of the structure and binding pockets of KRAS, medicinal chemistry optimization processes, and the biological characterization of small-molecule inhibitors that directly target KRAS, including covalent allosteric inhibitors specific for the G12C mutant, GTP-competitive inhibitors targeting the nucleotide-binding site, and protein-protein interaction inhibitors that bind in the switch I/II pocket or the A59 site. Additionally, we propose potential challenges faced by these new classes of KRAS inhibitors under clinical evaluation.

Although stress has been known to increase the susceptibility of pathogen infection, the underlying mechanism remains elusive. In this study, we reported that restraint stress dramatically enhanced the morbidity and mortality of mice infected with the influenza virus (H1N1) and obviously aggravated lung inflammation. Corticosterone (CORT), a main type of glucocorticoids in rodents, was secreted in the plasma of stressed mice. We further found that this stress hormone significantly boosted virus replication by restricting mitochondrial antiviral signaling (MAVS) protein-transduced IFN-β production without affecting its mRNA level, while the deficiency of MAVS abrogated stress/CORT-induced viral susceptibility in mice. Mechanistically, the effect of CORT was mediated by proteasome-dependent degradation of MAVS, thereby resulting in the impediment of MAVS-transduced IFN-β generation in vivo and in vitro. Furthermore, RNA-seq assay results indicated the involvement of Mitofusin 2 (Mfn2) in this process. Gain- and loss-of-function experiments indicated that Mfn2 interacted with MAVS and recruited E3 ligase SYVN1 to promote the polyubiquitination of MAVS. Co-immunoprecipitation experiments clarified an interaction between any two regions of Mfn2 (HR1), MAVS (C-terminal/TM) and SYVN1 (TM). Collectively, our findings define the Mfn2-SYVN1 axis as a new signaling cascade for proteasome-dependent degradation of MAVS and a ‘fine tuning’ of antiviral innate immunity in response to influenza infection under stress.

Extracellular matrix stiffness controls DNA damage response., DNA double-strand breaks (DSBs) are highly toxic lesions that can drive genetic instability. These lesions also contribute to the efficacy of radiotherapy and many cancer chemotherapeutics. DNA repair efficiency is regulated by both intracellular and extracellular chemical signals. However, it is largely unknown whether this process is regulated by physical stimuli such as extracellular mechanical signals. Here, we report that DSB repair is regulated by extracellular mechanical signals. Low extracellular matrix (ECM) stiffness impairs DSB repair and renders cells sensitive to genotoxic agents. Mechanistically, we found that the MAP4K4/6/7 kinases are activated and phosphorylate ubiquitin in cells at low stiffness. Phosphorylated ubiquitin impairs RNF8-mediated ubiquitin signaling at DSB sites, leading to DSB repair deficiency. Our results thus demonstrate that ECM stiffness regulates DSB repair efficiency and genotoxic sensitivity through MAP4K4/6/7 kinase–mediated ubiquitin phosphorylation, providing a previously unidentified regulation in DSB-induced ubiquitin signaling.

Triple-negative breast cancer (TNBC) is a heterogeneous disease with poor prognosis that lacks targeted therapies, especially in patients with chemotherapy-resistant disease. Since DNA methylation-induced silencing of tumor suppressors is common in cancer, reversal of promoter DNA hypermethylation by 5-aza-2'-deoxycytidine (decitabine), an FDA-approved DNA methyltransferase (DNMT) inhibitor, has proven effective in treating hematological neoplasms. However, its antitumor effect varies in solid tumors, stressing the importance of identifying biomarkers predictive of therapeutic response. Here, we focused on the identification of biomarkers to select decitabine-sensitive TNBC through increasing our understanding of the mechanism of decitabine action. We showed that protein levels of DNMTs correlated with response to decitabine in patient-derived xenograft (PDX) organoids originating from chemotherapy-sensitive and -resistant TNBCs, suggesting DNMT levels as potential biomarkers of response. Furthermore, all 3 methytransferases, DNMT1, DNMT3A, and DNMT3B, were degraded following low-concentration, long-term decitabine treatment both in vitro and in vivo. The DNMT proteins could be ubiquitinated by the E3 ligase, TNF receptor-associated factor 6 (TRAF6), leading to lysosome-dependent protein degradation. Depletion of TRAF6 blocked decitabine-induced DNMT degradation, conferring resistance to decitabine. Our study suggests a potential mechanism of regulating DNMT protein degradation and DNMT levels as response biomarkers for DNMT inhibitors in TNBCs.

Genomic stress leads to various forms of DNA damage, of which DNA double strand breaks (DSBs) are the most lethal. An army of signaling molecules is called to action as soon as these DNA breaks are detected. Various protein modifications, such as phosphorylation and ubiquitination, are an integral part of the reaction. While phosphorylation activates various proteins, ubiquitin (Ub) adducts typically act as docking sites for DNA repair factors. The response to DNA DSB starts with the protein kinase ATM phosphorylating various substrates including MDC1 and histone H2AX. This mediator protein, MDC1, then recognizes phosphorylated histone H2AX and amplifies the damage response. The E3 ligase, RNF8, recognizes and binds to phosphorylated MDC1. RNF8 then modifies an unknown protein to call a second ubiquitin ligase, RNF168, into action. It has been recognized that these two ubiquitin ligases are recruited sequentially but there is an unknown linker protein between them. These two ubiquitin ligases are crucial to the formation of DSB-associated ubiquitin conjugates and, as a result, there has been long standing interest in the field in identifying the link between the two factors. In this paper we identify lethal(3) malignant brain tumor like 2 (L3MBTL2) as the substrate of RNF8 (Nowsheen S, et al. Nat Cell Biol 20:455-464, 2018). We report that ATM-mediated phosphorylation of the polycomb group like protein L3MBTL2 and subsequent interaction with MDC1 brings it to the vicinity of the DNA lesion. RNF8 acts upon this phosphorylated L3MBTL2 and generates K63-linked polyubiquitin chains. This modified substrate is subsequently recognized by RNF168 and tethers the protein to the DNA lesion. RNF168 then ubiquitinates proteins such as histone H2A and H2AX to further amplify the damage response and recruit repair proteins such as BRCA1 and 53BP1 (Figure 1).

The melanocortin-1 receptor (MC1R), a G-protein-coupled receptor, has a crucial role in human and mouse pigmentation. Activation of MC1R in melanocytes by α-melanocyte-stimulating hormone (α-MSH) stimulates cAMP signalling and melanin production and enhances DNA repair after ultraviolet irradiation. Individuals carrying MC1R variants, especially those associated with red hair colour, fair skin and poor tanning ability (denoted as RHC variants), are associated with higher risk of melanoma. However, how MC1R activity is modulated by ultraviolet irradiation, why individuals with red hair are more prone to developing melanoma, and whether the activity of RHC variants might be restored for therapeutic benefit are unknown. Here we demonstrate a potential MC1R-targeted intervention strategy in mice to rescue loss-of-function MC1R in MC1R RHC variants for therapeutic benefit by activating MC1R protein palmitoylation. MC1R palmitoylation, primarily mediated by the protein-acyl transferase ZDHHC13, is essential for activating MC1R signalling, which triggers increased pigmentation, ultraviolet-B-induced G1-like cell cycle arrest and control of senescence and melanomagenesis in vitro and in vivo. Using C57BL/6J-Mc1re/eJ mice, in which endogenous MC1R is prematurely terminated, expressing Mc1r RHC variants, we show that pharmacological activation of palmitoylation rescues the defects of Mc1r RHC variants and prevents melanomagenesis. The results highlight a central role for MC1R palmitoylation in pigmentation and protection against melanoma.

Tumour metastasis, the spread of cancer cells from the original tumour site followed by growth of secondary tumours at distant organs, is the primary cause of cancer-related deaths and remains poorly understood. Here we demonstrate that inhibition of CDK4/6 blocks breast tumour metastasis in the triple-negative breast cancer model, without affecting tumour growth. Mechanistically, we identify a deubiquitinase, DUB3, as a target of CDK4/6; CDK4/6-mediated activation of DUB3 is essential to deubiquitinate and stabilize SNAIL1, a key factor promoting epithelial–mesenchymal transition and breast cancer metastasis. Overall, our study establishes the CDK4/6–DUB3 axis as an important regulatory mechanism of breast cancer metastasis and provides a rationale for potential therapeutic interventions in the treatment of breast cancer metastasis., Overexpression of SNAIL confers tumour cells with cancer stem-like characteristics associated with tumour progression. Here the authors show that inhibition of CDK4/6 blocks tumour metastasis in triple negative breast cancer by targeting DUB3 which in turns deubiquitinates and stabilises SNAIL1.

Summary Activating mutations in NRAS account for 20%–30% of melanoma, but despite decades of research and in contrast to BRAF, no effective anti-NRAS therapies have been forthcoming. Here, we identify a previously uncharacterized serine/threonine kinase STK19 as a novel NRAS activator. STK19 phosphorylates NRAS to enhance its binding to its downstream effectors and promotes oncogenic NRAS-mediated melanocyte malignant transformation. A recurrent D89N substitution in STK19 whose alterations were identified in 25% of human melanomas represents a gain-of-function mutation that interacts better with NRAS to enhance melanocyte transformation. STK19D89N knockin leads to skin hyperpigmentation and promotes NRASQ61R-driven melanomagenesis in vivo. Finally, we developed ZT-12-037-01 (1a) as a specific STK19-targeted inhibitor and showed that it effectively blocks oncogenic NRAS-driven melanocyte malignant transformation and melanoma growth in vitro and in vivo. Together, our findings provide a new and viable therapeutic strategy for melanomas harboring NRAS mutations.

The AMP-activated protein kinase (AMPK) is the master regulator of metabolic homeostasis by sensing cellular energy status. When intracellular ATP levels decrease during energy stress, AMPK is initially activated through AMP or ADP binding and phosphorylation of a threonine residue (Thr-172) within the activation loop of its kinase domain. Here we report a key molecular mechanism by which AMPK activation is amplified under energy stress. We found that ubiquitination on AMPKα blocks AMPKα phosphorylation by LKB1. The deubiquitinase USP10 specifically removes ubiquitination on AMPKα to facilitate AMPKα phosphorylation by LKB1. Under energy stress, USP10 activity in turn is enhanced through AMPK-mediated phosphorylation of Ser76 of USP10. Thus, USP10 and AMPK form a key feedforward loop ensuring amplification of AMPK activation in response to fluctuation of cellular energy status. Disruption of this feedforward loop leads to improper AMPK activation and multiple metabolic defects.

Elucidating mechanisms of chemoresistance is critical to improve cancer therapy, especially for the treatment of pancreatic ductal adenocarcinoma (PDAC). Genome-wide association studies have suggested the less studied gene HEAT repeat-containing protein 1 (HEATR1) as a possible determinant of cellular sensitivity to different chemotherapeutic drugs. In this study, we assessed this hypothesized link in PDAC, where HEATR1 expression is downregulated significantly. HEATR1 silencing in PDAC cells increased resistance to gemcitabine and other chemotherapeutics, where this effect was associated with increased AKT kinase phosphorylation at the Thr308 regulatory site. Mechanistically, HEATR1 enhanced cell responsiveness to gemcitabine by acting as a scaffold to facilitate interactions between AKT and the protein phosphatase PP2A, thereby promoting Thr308 dephosphorylation. Consistent with these findings, treatment with the AKT inhibitor triciribine sensitized HEATR1-depleted PDAC cells to gemcitabine, suggesting that this therapeutic combination may overcome gemcitabine resistance in patients with low HEATR1 expression. Clinically, we found that HEATR1 downregulation in PDAC patients was associated with increased AKT phosphorylation, poor response to tumor resection plus gemcitabine standard-of-care treatment, and shorter overall survival. Collectively, our findings establish HEATR1 as a novel regulator of AKT and a candidate predictive and prognostic indicator of drug responsiveness and outcome in PDAC patients. Cancer Res; 76(3); 572–81. ©2015 AACR.

Exosomes are attractive as nucleic-acid carriers because of their favourable pharmacokinetic and immunological properties and their ability to penetrate physiological barriers that are impermeable to synthetic drug-delivery vehicles. However, inserting exogenous nucleic acids, especially large messenger RNAs, into cell-secreted exosomes leads to low yields. Here we report a cellular-nanoporation method for the production of large quantities of exosomes containing therapeutic mRNAs and targeting peptides. We transfected various source cells with plasmid DNAs and stimulated the cells with a focal and transient electrical stimulus that promotes the release of exosomes carrying transcribed mRNAs and targeting peptides. Compared with bulk electroporation and other exosome-production strategies, cellular nanoporation produced up to 50-fold more exosomes and a more than 10

The ataxia-telangiectasia mutated (ATM) kinase, an upstream kinase of the DNA damage response (DDR), is rapidly activated following DNA damage, and phosphorylates its downstream targets to launch DDR signaling. However, the mechanism of ATM activation is still not completely understood. Here we report that UFM1 specific ligase 1 (UFL1), an ufmylation E3 ligase, is important for ATM activation. UFL1 is recruited to double strand breaks by the MRE11/RAD50/NBS1 complex, and monoufmylates histone H4 following DNA damage. Monoufmylated histone H4 is important for Suv39h1 and Tip60 recruitment. Furthermore, ATM phosphorylates UFL1 at serine 462, enhancing UFL1 E3 ligase activity and promoting ATM activation in a positive feedback loop. These findings reveal that ufmylation of histone H4 by UFL1 is an important step for amplification of ATM activation and maintenance of genomic integrity., Ufmylation is a ubiquitylation-like protein modification but only a few ufmylation substrates and functions have been discovered so far. Here, the authors demonstrate ufmylation of histone H4 upon DNA damage and show that this modification is involved in the amplification of ATM activation.

The von Hippel-Lindau tumor suppressor pVHL is an E3 ligase that targets hypoxia-inducible factors (HIFs). Mutation of VHL results in HIF up-regulation and contributes to processes related to tumor progression such as invasion, metastasis, and angiogenesis. However, very little is known with regard to post-transcriptional regulation of pVHL. Here we show that WD repeat and SOCS box-containing protein 1 (WSB1) is a negative regulator of pVHL through WSB1's E3 ligase activity. Mechanistically, WSB1 promotes pVHL ubiquitination and proteasomal degradation, thereby stabilizing HIF under both normoxic and hypoxic conditions. As a consequence, WSB1 up-regulates the expression of HIF-1α’s target genes and promotes cancer invasion and metastasis through its effect on pVHL. Consistent with this, WSB1 protein level negatively correlates with pVHL level and metastasis-free survival in clinical samples. This work reveals a new mechanism of pVHL's regulation by which cancer acquires invasiveness and metastatic tendency.