Molecular insights and clinical implications for the tumor suppressor role of SCFFBXW7 E3 ubiquitin ligase.

FBXW7 is one of the most well-characterized F-box proteins, serving as substrate receptor subunit of SKP1-CUL1-F-box (SCF) E3 ligase complexes. SCFFBXW7 is responsible for the degradation of various oncogenic proteins such as cyclin E, c-MYC, c-JUN, NOTCH, and MCL1. Therefore, FBXW7 functions largely as a major tumor suppressor. In keeping with this notion, FBXW7 gene mutations or downregulations have been found and reported in many types of malignant tumors, such as endometrial, colorectal, lung, and breast cancers, which facilitate the proliferation, invasion, migration, and drug resistance of cancer cells. Therefore, it is critical to review newly identified FBXW7 regulation and tumor suppressor function under physiological and pathological conditions to develop effective strategies for the treatment of FBXW7-altered cancers. Since a growing body of evidence has revealed the tumor-suppressive activity and role of FBXW7, here, we updated FBXW7 upstream and downstream signaling including FBXW7 ubiquitin substrates, the multi-level FBXW7 regulatory mechanisms, and dysregulation of FBXW7 in cancer, and discussed promising cancer therapies targeting FBXW7 regulators and downstream effectors, to provide a comprehensive picture of FBXW7 and facilitate the study in this field.

USP2 inhibition prevents infection with ACE2-dependent coronaviruses in vitro and is protective against SARS-CoV-2 in mice.

Targeting angiotensin-converting enzyme 2 (ACE2) represents a promising and effective approach to combat not only the COVID-19 pandemic but also potential future pandemics arising from coronaviruses that depend on ACE2 for infection. Here, we report ubiquitin specific peptidase 2 (USP2) as a host-directed antiviral target; we further describe the development of MS102, an orally available USP2 inhibitor with viable antiviral activity against ACE2-dependent coronaviruses. Mechanistically, USP2 serves as a physiological deubiquitinase of ACE2, and targeted inhibition with specific small-molecule inhibitor ML364 leads to a marked and reversible reduction in ACE2 protein abundance, thereby blocking various ACE2-dependent coronaviruses tested. Using human ACE2 transgenic mouse models, we further demonstrate that ML364 efficiently controls disease caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), as evidenced by reduced viral loads and ameliorated lung inflammation. Furthermore, we improved the in vivo performance of ML364 in terms of both pharmacokinetics and antiviral activity. The resulting lead compound, MS102, holds promise as an oral therapeutic option for treating infections with coronaviruses that are reliant on ACE2.

The circadian clock, aging and its implications in cancer.

Circadian clock orchestrates the intergenic biochemical, physiological and behavioral changes to form an approximate 24h oscillation through the transcription-translation feedback loop (TTFL). Mechanistically, a heterodimer of transcriptional activator formed by BMAL1 and CLOCK, governs the expression of its transcriptional repressors, CRY, PER and REV-ERBα/ß proteins, thereby controlling more than 50 % of protein encoding genes in human. There is also increasing evidence showing that tumor microenvironment can disrupt specific clock gene functions to facilitate tumorigenesis. Although there is great progress in understanding the molecular mechanisms of the circadian clock, aging and cancer, elucidating their complex relationships among these processes remains challenging. Herein, the optimization of the chronochemotherapy regimen has not been justified yet for treatment of cancer. Here, we discuss the hypothesis of relocalization of chromatin modifiers (RCM) along with function(s) of the circadian rhythm on aging and carcinogenesis. We will also introduce the function of the chromatin remodeling as a new avenue for rejuvenation of competent tissues to combat aging and cancer.

Insights into the aberrant CDK4/6 signaling pathway as a therapeutic target in tumorigenesis.

The recent findings advance our knowledge for the prevention of the premature activation of the major oncogenic pathways including MYC and the cyclin D-cyclin-dependent kinases 4 and 6 (CDK4/6) axis. D-type cyclins are frequently deregulated in human cancer and promote cell division in part through activation of CDK4/6. Therefore, the activation of the cyclin D-CDK4/6 axis stimulates cell proliferation and cancer progression, which represents a unique therapeutic target. However, we have shown that inhibition of CDK4/6 upregulates protein levels of RB1 and CDK6 for acquisition of drug resistance to CDK4/6 inhibitors. Here, we review new progress in the control of cyclin D-dependent cancer cell cycle and proliferation, along with identification of novel E3 ligase for the stability of cyclin D. Cullin4-RING E3 ligase (CRL4)AMBRA1 complex plays a critical role in regulating D-type cyclins through their protein destabilization to control S phase entry and maintain genomic integrity. We also summarize the strategy for inhibition of the cyclin D-associated kinases CDK4/6 and other potential cell cycle regulators for targeting cancer with altered cyclin D expression. We also uncover the function of CK1ɛ as an effective target to potentiate therapeutic efficacy of CDK4/6 inhibitors. Moreover, as the level of PD-L1 is considered in the severe clinical problem in the patients treated with CDK4 inhibitors, we assume that a therapeutic combination using PD-L1 immunotherapy might lower the development of drug resistance and targeting cyclin D will likely inhibit tumor growth and overcome resistance to cyclin D-associated CDK4/6 inhibitors.

Pharmacological inhibition of the SKP2/p300 signaling axis restricts castration-resistant prostate cancer.

SKP2, an F-box protein of the SCF type of the E3 ubiquitin ligase complex, plays an important function in driving tumorigenesis through the destruction of numerous tumor-suppressive proteins. Besides its critical role in cell cycle regulation, proto-oncogenic functions of SKP2 have also been shown in a cell cycle regulation-independent manner. Therefore, uncovering novel physiological upstream regulators of SKP2 signaling pathways would be essential to retard aggressive malignancies. Here, we report that elevation of SKP2 and EP300 transcriptomic expression is a hallmark of castration-resistant prostate cancer. We also found that SKP2 acetylation is likely a critical driven event in castration-resistant prostate cancer cells. Mechanistically, SKP2-acetylation is mediated by the p300 acetyltransferase enzyme for post-translational modification (PTM) event that is induced upon stimulation with dihydrotestosterone (DHT) in prostate cancer cells. Moreover, ectopic expression of acetylation-mimetic K68/71Q mutant of SKP2 in LNCaP cells could confer resistance to androgen withdrawal-induced growth arrest and promotes prostate cancer stem cell (CSC)-like traits including survival, proliferation, stemness formation, lactate production, migration, and invasion. Furthermore, inhibition of p300-mediated SKP2 acetylation or SKP2-mediated p27-degradation by pharmacological inhibition of p300 or SKP2 could attenuate epithelial-mesenchymal transition (EMT) and the proto-oncogenic activities of the SKP2/p300 and androgen receptor (AR) signaling pathways. Therefore, our study identifies the SKP2/p300 axis as a possible molecular mechanism driving castration-resistant prostate cancers, which provides pharmaceutical insight into inactivation of the SKP2/p300 axis for restriction of CSC-like properties, thereby benefiting clinical diagnosis and cancer therapy.

Transcriptome, proteome, and protein synthesis within the intracellular cytomatrix.

Despite the knowledge that protein translation and various metabolic reactions that create and sustain cellular life occur in the cytoplasm, the structural organization within the cytoplasm remains unclear. Recent models indicate that cytoplasm contains viscous fluid and elastic solid phases. We separated these viscous fluid and solid elastic compartments, which we call the cytosol and cytomatrix, respectively. The distinctive composition of the cytomatrix included structural proteins, ribosomes, and metabolome enzymes. High-throughput analysis revealed unique biosynthetic pathways within the cytomatrix. Enrichment of biosynthetic pathways in the cytomatrix indicated the presence of immobilized biocatalysis. Enzymatic immobilization and segregation can surmount spatial impediments, and the local pathway segregation may form cytoplasmic organelles. Protein translation was reprogrammed within the cytomatrix under the restriction of protein synthesis by drug treatment. The cytosol and cytomatrix are an elaborately interconnected network that promotes operational flexibility in healthy cells and the survival of malignant cells.

PROTACs technology for treatment of Alzheimer's disease: Advances and perspectives.

Neurodegenerative diseases (NDs) are characteristic with progression of neuron degeneration, resulting in dysfunction of cognition and mobility. Many neurodegenerative diseases are because of proteinopathies that results from unusual protein accumulations and aggregations. The aggregation of misfolded proteins like ß-amyloid, α-synuclein, tau, and polyglutamates are hallmarked in Alzheimer's disease (AD), which are undruggable targets, and usually do not respond to conventional small-molecule agents. Therefore, developing novel technology and strategy for reducing the levels of protein aggregates would be critical for treatment of AD. Recently, the emerging proteolysis targeting chimeras (PRPTACs) technology has been significantly considered for artificial and selective degradation of aberrant target proteins. These engineered bifunctional molecules engage target proteins to be degraded by either the cellular degradation machinery in the ubiquitin-proteasome system (UPS) or via the autophagy-lysosome degradation pathway. Although the application of PROTACs technology is preferable than oligonucleotide and antibodies for treatment of NDs, many limitations such as their pharmacokinetic properties, tissue distribution and cell permeabilities, still need to be corrected. Herein, we review the recent advances in PROTACs technology with their limitation for pharmaceutical targeting of aberrant proteins involved in Alzheimer's diseases. We also review therapeutic potential of dysregulated signaling such as PI3K/AKT/mTOR axis for the management of AD.

The regulation of neuronal autophagy and cell survival by MCL1 in Alzheimer's disease.

Maintaining neuronal integrity and functions requires precise mechanisms controlling organelle and protein quality. Alzheimer's disease (AD) is characterized by functional defects in the clearance and recycling of intracellular components. As such, neuronal homeostasis involves autophagy, mitophagy, and apoptosis. Compromised activity in these cellular processes may cause pathological phenotypes of AD. Dysfunction of mitochondria is one of the hallmarks of AD. Mitophagy is a critical mitochondria quality control system, and the impaired mitophagy is observed in AD. Myeloid cell leukemia 1 (MCL1), a member of the pro-survival B-cell lymphoma protein 2 (BCL2) family, is a mitochondria-targeted protein that contributes to maintaining mitochondrial integrity. Mcl1 knockout mice display peri-implantation lethality. The studies on conditional Mcl1 knockout mice demonstrate that MCL1 plays a central role in neurogenesis and neuronal survival during brain development. Accumulating evidence reveals the critical role of MCL1 as a regulator of neuronal autophagy, mitophagy, and survival. In this review, we discuss the emerging neuroprotective function of MCL1 and how dysregulation of MCL1 signaling is involved in the pathogenesis of AD. As the pro-survival BCL2 family of proteins are promising targets of pharmacological intervention with BH3 mimetic drugs, we also discuss the promise of MCL1-targeting therapy in AD.

ATM: Main Features, Signaling Pathways, and Its Diverse Roles in DNA Damage Response, Tumor Suppression, and Cancer Development.

ATM is among of the most critical initiators and coordinators of the DNA-damage response. ATM canonical and non-canonical signaling pathways involve hundreds of downstream targets that control many important cellular processes such as DNA damage repair, apoptosis, cell cycle arrest, metabolism, proliferation, oxidative sensing, among others. Of note, ATM is often considered a major tumor suppressor because of its ability to induce apoptosis and cell cycle arrest. However, in some advanced stage tumor cells, ATM signaling is increased and confers remarkable advantages for cancer cell survival, resistance to radiation and chemotherapy, biosynthesis, proliferation, and metastasis. This review focuses on addressing major characteristics, signaling pathways and especially the diverse roles of ATM in cellular homeostasis and cancer development.

Phosphorylation of PDHA by AMPK Drives TCA Cycle to Promote Cancer Metastasis.

Cancer metastasis accounts for the major cause of cancer-related deaths. How disseminated cancer cells cope with hostile microenvironments in secondary site for full-blown metastasis is largely unknown. Here, we show that AMPK (AMP-activated protein kinase), activated in mouse metastasis models, drives pyruvate dehydrogenase complex (PDHc) activation to maintain TCA cycle (tricarboxylic acid cycle) and promotes cancer metastasis by adapting cancer cells to metabolic and oxidative stresses. This AMPK-PDHc axis is activated in advanced breast cancer and predicts poor metastasis-free survival. Mechanistically, AMPK localizes in the mitochondrial matrix and phosphorylates the catalytic alpha subunit of PDHc (PDHA) on two residues S295 and S314, which activates the enzymatic activity of PDHc and alleviates an inhibitory phosphorylation by PDHKs, respectively. Importantly, these phosphorylation events mediate PDHc function in cancer metastasis. Our study reveals that AMPK-mediated PDHA phosphorylation drives PDHc activation and TCA cycle to empower cancer cells adaptation to metastatic microenvironments for metastasis.

Author Correction: A hypoxia-responsive TRAF6-ATM-H2AX signalling axis promotes HIF1α activation, tumorigenesis and metastasis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Therapeutic Potential of the miRNA-ATM Axis in the Management of Tumor Radioresistance.

The ataxia-telangiectasia mutated (ATM) protein kinase is widely known for its function as a chief mobilizer of the DNA damage response (DDR) upon DNA double-strand breaks. ATM orchestrates the DDR by modulating the expression of various miRNAs through several mechanisms. On the other hand, a set of miRNAs contribute to tight regulation of ATM by directly targeting the 3'-untranslated region of ATM mRNA. This review addresses the therapeutic application and molecular mechanisms that underlie the intricate interactions between miRNAs and ATM. It also describes therapeutic delivery of miRNAs in different environments such as hypoxic tumor microenvironments.

Atad3a suppresses Pink1-dependent mitophagy to maintain homeostasis of hematopoietic progenitor cells.

Although deletion of certain autophagy-related genes has been associated with defects in hematopoiesis, it remains unclear whether hyperactivated mitophagy affects the maintenance and differentiation of hematopoietic stem cells (HSCs) and committed progenitor cells. Here we report that targeted deletion of the gene encoding the AAA+-ATPase Atad3a hyperactivated mitophagy in mouse hematopoietic cells. Affected mice showed reduced survival, severely decreased bone-marrow cellularity, erythroid anemia and B cell lymphopenia. Those phenotypes were associated with skewed differentiation of stem and progenitor cells and an enlarged HSC pool. Mechanistically, Atad3a interacted with the mitochondrial channel components Tom40 and Tim23 and served as a bridging factor to facilitate appropriate transportation and processing of the mitophagy protein Pink1. Loss of Atad3a caused accumulation of Pink1 and activated mitophagy. Notably, deletion of Pink1 in Atad3a-deficient mice significantly 'rescued' the mitophagy defect, which resulted in restoration of the progenitor and HSC pools. Our data indicate that Atad3a suppresses Pink1-dependent mitophagy and thereby serves a key role in hematopoietic homeostasis.

H3 ubiquitination by NEDD4 regulates H3 acetylation and tumorigenesis.

Dynamic changes in histone modifications under various physiological cues play important roles in gene transcription and cancer. Identification of new histone marks critical for cancer development is of particular importance. Here we show that, in a glucose-dependent manner, E3 ubiquitin ligase NEDD4 ubiquitinates histone H3 on lysine 23/36/37 residues, which specifically recruits histone acetyltransferase GCN5 for subsequent H3 acetylation. Genome-wide analysis of chromatin immunoprecipitation followed by sequencing reveals that NEDD4 regulates glucose-induced H3 K9 acetylation at transcription starting site and enhancer regions. Integrative analysis of ChIP-seq and microarray data sets also reveals a consistent role of NEDD4 in transcription activation and H3 K9 acetylation in response to glucose. Functionally, we show that NEDD4-mediated H3 ubiquitination, by transcriptionally activating IL1α, IL1ß and GCLM, is important for tumour sphere formation. Together, our study reveals the mechanism for glucose-induced transcriptome reprograming and epigenetic regulation in cancer by inducing NEDD4-dependent H3 ubiquitination.

A hypoxia-responsive TRAF6-ATM-H2AX signalling axis promotes HIF1α activation, tumorigenesis and metastasis.

The understanding of how hypoxia stabilizes and activates HIF1α in the nucleus with related oncogenic signals could revolutionize targeted therapy for cancers. Here, we find that histone H2AX displays oncogenic activity by serving as a crucial regulator of HIF1α signalling. H2AX interacts with HIF1α to prevent its degradation and nuclear export in order to allow successful VHL-independent HIF1α transcriptional activation. We show that mono-ubiquitylation and phosphorylation of H2AX, which are strictly mediated by hypoxia-induced E3 ligase activity of TRAF6 and ATM, critically regulate HIF1α-driven tumorigenesis. Importantly, TRAF6 and γH2AX are overexpressed in human breast cancer, correlate with activation of HIF1α signalling, and predict metastatic outcome. Thus, TRAF6 and H2AX overexpression and γH2AX-mediated HIF1α enrichment in the nucleus of cancer cells lead to overactivation of HIF1α-driven tumorigenesis, glycolysis and metastasis. Our findings suggest that TRAF6-mediated mono-ubiquitylation and subsequent phosphorylation of H2AX may serve as potential means for cancer diagnosis and therapy.

TRAF6 Restricts p53 Mitochondrial Translocation, Apoptosis, and Tumor Suppression.

Mitochondrial p53 is involved in apoptosis and tumor suppression. However, its regulation is not well studied. Here, we show that TRAF6 E3 ligase is a crucial factor to restrict mitochondrial translocation of p53 and spontaneous apoptosis by promoting K63-linked ubiquitination of p53 at K24 in cytosol, and such ubiquitination limits the interaction between p53 and MCL-1/BAK. Genotoxic stress reduces this ubiquitination in cytosol by S13/T330 phosphorylation-dependent translocation of TRAF6 from cytosol to nucleus, where TRAF6 also facilitates the K63-linked ubiquitination of nuclear p53 and its transactivation by recruiting p300 for p53 acetylation. Functionally, K63-linked ubiquitination of p53 compromised p53-mediated apoptosis and tumor suppression. Colorectal cancer samples with WT p53 reveal that TRAF6 overexpression negatively correlates with apoptosis and predicts poor response to chemotherapy and radiotherapy. Together, our study identifies TRAF6 as a critical gatekeeper to restrict p53 mitochondrial translocation, and such mechanism may contribute to tumor development and drug resistance.

Skp2-Mediated RagA Ubiquitination Elicits a Negative Feedback to Prevent Amino-Acid-Dependent mTORC1 Hyperactivation by Recruiting GATOR1.

The regulation of RagA(GTP) is important for amino-acid-induced mTORC1 activation. Although GATOR1 complex has been identified as a negative regulator for mTORC1 by hydrolyzing RagA(GTP), how GATOR1 is recruited to RagA to attenuate mTORC1 signaling remains unclear. Moreover, how mTORC1 signaling is terminated upon amino acid stimulation is also unknown. We show that the recruitment of GATOR1 to RagA is induced by amino acids in an mTORC1-dependent manner. Skp2 E3 ligase drives K63-linked ubiquitination of RagA, which facilitates GATOR1 recruitment and RagA(GTP) hydrolysis, thereby providing a negative feedback loop to attenuate mTORC1 lysosomal recruitment and prevent mTORC1 hyperactivation. We further demonstrate that Skp2 promotes autophagy but inhibits cell size and cilia growth through RagA ubiquitination and mTORC1 inhibition. We thereby propose a negative feedback whereby Skp2-mediated RagA ubiquitination recruits GATOR1 to restrict mTORC1 signaling upon sustained amino acid stimulation, which serves a critical mechanism to maintain proper cellular functions.

Skp2-macroH2A1-CDK8 axis orchestrates G2/M transition and tumorigenesis.

Understanding the mechanism by which cell growth, migration, polyploidy, and tumorigenesis are regulated may provide important therapeutic strategies for cancer therapy. Here we identify the Skp2-macroH2A1 (mH2A1)-cyclin-dependent kinase 8 (CDK8) axis as a critical pathway for these processes, and deregulation of this pathway is associated with human breast cancer progression and patient survival outcome. We showed that mH2A1 is a new substrate of Skp2 SCF complex whose degradation by Skp2 promotes CDK8 gene and protein expression. Strikingly, breast tumour suppression on Skp2 deficiency can be rescued by mH2A1 knockdown or CDK8 restoration using mouse tumour models. We further show that CDK8 regulates p27 protein expression by facilitating Skp2-mediated p27 ubiquitination and degradation. Our study establishes a critical role of Skp2-mH2A1-CDK8 axis in breast cancer development and targeting this pathway offers a promising strategy for breast cancer therapy.

Skp2-dependent ubiquitination and activation of LKB1 is essential for cancer cell survival under energy stress.

LKB1 is activated by forming a heterotrimeric complex with STRAD and MO25. Recent studies suggest that LKB1 has pro-oncogenic functions, besides acting as a tumor suppressor. How the LKB1 activity is maintained and how LKB1 regulates cancer development are largely unclear. Here we show that K63-linked LKB1 polyubiquitination by Skp2-SCF ubiquitin ligase is critical for LKB1 activation by maintaining LKB1-STRAD-MO25 complex integrity. We further demonstrate that oncogenic Ras acts upstream of Skp2 to promote LKB1 polyubiquitination by activating Skp2-SCF ubiquitin ligase. Moreover, Skp2-mediated LKB1 polyubiquitination is required for energy-stress-induced cell survival. We also detected overexpression of Skp2 and LKB1 in late-stage hepatocellular carcinoma (HCC), and their overexpression predicts poor survival outcomes. Finally, we show that Skp2-mediated LKB1 polyubiquitination is important for HCC tumor growth in vivo. Our study provides new insights into the upstream regulation of LKB1 activation and suggests a potential target, the Ras/Skp2/LKB1 axis, for cancer therapy.