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Molecular Cell Oct 2020Cancer metastasis accounts for the major cause of cancer-related deaths. How disseminated cancer cells cope with hostile microenvironments in secondary site for...
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.
Topics: AMP-Activated Protein Kinases; Animals; Breast Neoplasms; Catalytic Domain; Cell Line, Tumor; Cell Survival; Citric Acid Cycle; Enzyme Activation; Female; Humans; Mice, Inbred BALB C; Mice, Nude; Neoplasm Metastasis; Phosphorylation; Phosphoserine; Pyruvate Dehydrogenase Complex; Signal Transduction; Stress, Physiological; Survival Analysis
PubMed: 33022274
DOI: 10.1016/j.molcel.2020.09.018 -
Nature Jan 2023Protein phosphorylation is one of the most widespread post-translational modifications in biology. With advances in mass-spectrometry-based phosphoproteomics, 90,000...
Protein phosphorylation is one of the most widespread post-translational modifications in biology. With advances in mass-spectrometry-based phosphoproteomics, 90,000 sites of serine and threonine phosphorylation have so far been identified, and several thousand have been associated with human diseases and biological processes. For the vast majority of phosphorylation events, it is not yet known which of the more than 300 protein serine/threonine (Ser/Thr) kinases encoded in the human genome are responsible. Here we used synthetic peptide libraries to profile the substrate sequence specificity of 303 Ser/Thr kinases, comprising more than 84% of those predicted to be active in humans. Viewed in its entirety, the substrate specificity of the kinome was substantially more diverse than expected and was driven extensively by negative selectivity. We used our kinome-wide dataset to computationally annotate and identify the kinases capable of phosphorylating every reported phosphorylation site in the human Ser/Thr phosphoproteome. For the small minority of phosphosites for which the putative protein kinases involved have been previously reported, our predictions were in excellent agreement. When this approach was applied to examine the signalling response of tissues and cell lines to hormones, growth factors, targeted inhibitors and environmental or genetic perturbations, it revealed unexpected insights into pathway complexity and compensation. Overall, these studies reveal the intrinsic substrate specificity of the human Ser/Thr kinome, illuminate cellular signalling responses and provide a resource to link phosphorylation events to biological pathways.
Topics: Humans; Phosphorylation; Protein Serine-Threonine Kinases; Serine; Substrate Specificity; Threonine; Proteome; Datasets as Topic; Phosphoproteins; Cell Line; Phosphoserine; Phosphothreonine
PubMed: 36631611
DOI: 10.1038/s41586-022-05575-3 -
Cell Death & Disease Jan 2020Excessive mitochondrial fission acts as a pro-proliferative marker in some cancers and organ fibrosis; its potential role in renal fibroblast activation and fibrogenesis...
Excessive mitochondrial fission acts as a pro-proliferative marker in some cancers and organ fibrosis; its potential role in renal fibroblast activation and fibrogenesis has never been investigated. Here, we showed more pronounced fragmented mitochondria in fibrotic than in non-fibrotic renal fibroblast in humans and mice. In a mouse model of obstructive nephropathy, phosphorylation of Drp1 at serine 616 (p-Drp1S616) and acetylation of H3K27(H3K27ac) was increased in fibrotic kidneys; pharmacological inhibition of mitochondrial fission by mdivi-1 substantially reduced H3K27ac levels, fibroblasts accumulation, and interstitial fibrosis. Moreover, mdivi-1 treatment was able to attenuate the established renal fibrosis. In cultured renal interstitial fibroblasts, targeting Drp1 using pharmacological inhibitor or siRNA suppressed TGF-β1-elicited cell activation and proliferation, as evidenced by inhibiting expression of α-smooth muscle actin (α-SMA) and collagen I, as well as by reducing DNA synthesis. In contrast, Drp1 deletion enhanced cell apoptosis, along with decreased mitochondrial fragmentation, mtROS elevation, and glycolytic shift upon TGF-β1 stimulation. In Drp1 deletion fibroblasts, re-expression of wild-type Drp1 rather than Drp1S616A mutant restores the reduction of TGF-β-induced-Drp1 phosphorylation, H3K27ac, and cell activation. Moreover, TGF-β1 treatment increased the enrichment of H3K27ac at the promoters of α-SMA and PCNA, which was reversed in Drp1-knockdown fibroblasts co-transfected with empty vector or Drp1S616A, but not wild-type Drp1. Collectively, our results imply that inhibiting p-Drp1S616-mediated mitochondrial fission attenuates fibroblast activation and proliferation in renal fibrosis through epigenetic regulation of fibrosis-related genes transcription and may serve as a therapeutic target for retarding progression of chronic kidney disease.
Topics: Animals; Apoptosis; Cell Proliferation; Dynamins; Fibroblasts; Fibrosis; Gene Knockdown Techniques; Histones; Humans; Kidney; Lysine; Male; Mice, Inbred C57BL; Mitochondria; Mitochondrial Dynamics; Phosphorylation; Phosphoserine; Proliferating Cell Nuclear Antigen; Promoter Regions, Genetic; Protein Binding; Rats; Reactive Oxygen Species; Transforming Growth Factor beta1
PubMed: 31949126
DOI: 10.1038/s41419-019-2218-5 -
Molecular Cancer Dec 2020N6-methyladenosine (m6A) is the most prevalent messenger RNA modification in mammalian cells. However, the disease relevant function of m6A on specific oncogenic long...
BACKGROUND
N6-methyladenosine (m6A) is the most prevalent messenger RNA modification in mammalian cells. However, the disease relevant function of m6A on specific oncogenic long non-coding RNAs (ncRNAs) is not well understood.
METHODS
We analyzed the m6A status using patients samples and bone metastatic PDXs. Through m6A high-throughput sequencing, we identified the m6A sites on NEAT1-1 in prostate bone metastatic PDXs. Mass spec assay showed interaction among NEAT1-1, CYCLINL1 and CDK19. RNA EMSA, RNA pull-down, mutagenesis, CLIP, western blot, ChIP and ChIRP assays were used to investigate the molecular mechanisms underlying the functions of m6A on NEAT1-1. Loss-of function and rescued experiments were executed to detect the biological roles of m6A on NEAT1-1 in the PDX cell phenotypes in vivo.
RESULTS
In this study, we identified 4 credible m6A sites on long ncRNA NEAT1-1. High m6A level of NEAT1-1 was related to bone metastasis of prostate cancer and m6A level of NEAT1-1 was a powerful predictor of eventual death. Transcribed NEAT1-1 served as a bridge to facility the binding between CYCLINL1 and CDK19 and promoted the Pol II ser2 phosphorylation. Importantly, depletion of NEAT1-1or decreased m6A of NEAT1-1 impaired Pol II Ser-2p level in the promoter of RUNX2. Overexpression of NEAT1-1 induced cancer cell metastasis to lung and bone; xenograft growth and shortened the survival of mice, but NEAT1-1 with m6A site mutation failed to do these.
CONCLUSION
Collectively, the findings indicate that m6A on ncRNA NEAT1-1 takes critical role in regulating Pol II ser2 phosphorylation and may be novel specific target for bone metastasis cancer therapy and diagnosis. New complex CYCLINL1/CDK19/NEAT1-1 might provide new insight into the potential mechanism of the pathogenesis and development of bone metastatic prostate cancer.
Topics: Adenosine; Aged; Aged, 80 and over; Animals; Bone Neoplasms; Cell Line, Tumor; Core Binding Factor Alpha 1 Subunit; Cyclin-Dependent Kinases; Cyclins; Humans; Male; Mice, Nude; Middle Aged; Phosphorylation; Phosphoserine; Prognosis; Promoter Regions, Genetic; Prostatic Neoplasms; RNA Polymerase II; RNA, Long Noncoding
PubMed: 33308223
DOI: 10.1186/s12943-020-01293-4 -
Nature Jan 2016Triple-negative breast cancer (TNBC) is a heterogeneous and clinically aggressive disease for which there is no targeted therapy. BET bromodomain inhibitors, which have...
Triple-negative breast cancer (TNBC) is a heterogeneous and clinically aggressive disease for which there is no targeted therapy. BET bromodomain inhibitors, which have shown efficacy in several models of cancer, have not been evaluated in TNBC. These inhibitors displace BET bromodomain proteins such as BRD4 from chromatin by competing with their acetyl-lysine recognition modules, leading to inhibition of oncogenic transcriptional programs. Here we report the preferential sensitivity of TNBCs to BET bromodomain inhibition in vitro and in vivo, establishing a rationale for clinical investigation and further motivation to understand mechanisms of resistance. In paired cell lines selected for acquired resistance to BET inhibition from previously sensitive TNBCs, we failed to identify gatekeeper mutations, new driver events or drug pump activation. BET-resistant TNBC cells remain dependent on wild-type BRD4, which supports transcription and cell proliferation in a bromodomain-independent manner. Proteomic studies of resistant TNBC identify strong association with MED1 and hyper-phosphorylation of BRD4 attributable to decreased activity of PP2A, identified here as a principal BRD4 serine phosphatase. Together, these studies provide a rationale for BET inhibition in TNBC and present mechanism-based combination strategies to anticipate clinical drug resistance.
Topics: Animals; Azepines; Binding, Competitive; Casein Kinase II; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Chromatin; Drug Resistance, Neoplasm; Epigenesis, Genetic; Female; Gene Expression Regulation, Neoplastic; Genome, Human; Humans; Mediator Complex Subunit 1; Mice; Nuclear Proteins; Phosphorylation; Phosphoserine; Protein Binding; Protein Phosphatase 2; Protein Structure, Tertiary; Proteomics; Transcription Factors; Transcription, Genetic; Triazoles; Triple Negative Breast Neoplasms; Xenograft Model Antitumor Assays
PubMed: 26735014
DOI: 10.1038/nature16508 -
Molecular Plant Feb 2021Low temperature is a major environmental factor that limits plant growth and productivity. Although transient elevation of cytoplasmic calcium has long been recognized...
Low temperature is a major environmental factor that limits plant growth and productivity. Although transient elevation of cytoplasmic calcium has long been recognized as a critical signal for plant cold tolerance, the calcium channels responsible for this process have remained largely elusive. Here we report that OsCNGC9, a cyclic nucleotide-gated channel, positively regulates chilling tolerance by mediating cytoplasmic calcium elevation in rice (Oryza sativa). We showed that the loss-of-function mutant of OsCNGC9 is defective in cold-induced calcium influx and more sensitive to prolonged cold treatment, whereas OsCNGC9 overexpression confers enhanced cold tolerance. Mechanistically, we demonstrated that in response to chilling stress, OsSAPK8, a homolog of Arabidopsis thaliana OST1, phosphorylates and activates OsCNGC9 to trigger Ca influx. Moreover, we found that the transcription of OsCNGC9 is activated by a rice dehydration-responsive element-binding transcription factor, OsDREB1A. Taken together, our results suggest that OsCNGC9 enhances chilling tolerance in rice through regulating cold-induced calcium influx and cytoplasmic calcium elevation.
Topics: Adaptation, Physiological; Amino Acid Sequence; Calcium; Calcium Channels; Cold Temperature; Gene Expression Regulation, Plant; Models, Biological; Mutation; Nucleotide Motifs; Oryza; Phosphorylation; Phosphoserine; Plant Proteins; Promoter Regions, Genetic; Protein Binding; Response Elements; Seedlings; Stress, Physiological; Transcription, Genetic; Transcriptional Activation
PubMed: 33278597
DOI: 10.1016/j.molp.2020.11.022 -
Nature Jul 2018Diabetes is a complex metabolic syndrome that is characterized by prolonged high blood glucose levels and frequently associated with life-threatening complications....
Diabetes is a complex metabolic syndrome that is characterized by prolonged high blood glucose levels and frequently associated with life-threatening complications. Epidemiological studies have suggested that diabetes is also linked to an increased risk of cancer. High glucose levels may be a prevailing factor that contributes to the link between diabetes and cancer, but little is known about the molecular basis of this link and how the high glucose state may drive genetic and/or epigenetic alterations that result in a cancer phenotype. Here we show that hyperglycaemic conditions have an adverse effect on the DNA 5-hydroxymethylome. We identify the tumour suppressor TET2 as a substrate of the AMP-activated kinase (AMPK), which phosphorylates TET2 at serine 99, thereby stabilizing the tumour suppressor. Increased glucose levels impede AMPK-mediated phosphorylation at serine 99, which results in the destabilization of TET2 followed by dysregulation of both 5-hydroxymethylcytosine (5hmC) and the tumour suppressive function of TET2 in vitro and in vivo. Treatment with the anti-diabetic drug metformin protects AMPK-mediated phosphorylation of serine 99, thereby increasing TET2 stability and 5hmC levels. These findings define a novel 'phospho-switch' that regulates TET2 stability and a regulatory pathway that links glucose and AMPK to TET2 and 5hmC, which connects diabetes to cancer. Our data also unravel an epigenetic pathway by which metformin mediates tumour suppression. Thus, this study presents a new model for how a pernicious environment can directly reprogram the epigenome towards an oncogenic state, offering a potential strategy for cancer prevention and treatment.
Topics: 5-Methylcytosine; Adenylate Kinase; Animals; DNA; DNA Methylation; DNA-Binding Proteins; Diabetes Mellitus; Dioxygenases; Enzyme Stability; Epigenesis, Genetic; Glucose; Glycated Hemoglobin; Humans; Hyperglycemia; Metformin; Mice; Mice, Nude; Neoplasms; Phosphorylation; Phosphoserine; Proto-Oncogene Proteins; Substrate Specificity; Xenograft Model Antitumor Assays
PubMed: 30022161
DOI: 10.1038/s41586-018-0350-5 -
Nature Communications Jul 2021Despite the substantial impact of post-translational modifications on programmed cell death 1 ligand 1 (PD-L1), its importance in therapeutic resistance in pancreatic...
Despite the substantial impact of post-translational modifications on programmed cell death 1 ligand 1 (PD-L1), its importance in therapeutic resistance in pancreatic cancer remains poorly defined. Here, we demonstrate that never in mitosis gene A-related kinase 2 (NEK2) phosphorylates PD-L1 to maintain its stability, causing PD-L1-targeted pancreatic cancer immunotherapy to have poor efficacy. We identify NEK2 as a prognostic factor in immunologically "hot" pancreatic cancer, involved in the onset and development of pancreatic tumors in an immune-dependent manner. NEK2 deficiency results in the suppression of PD-L1 expression and enhancement of lymphocyte infiltration. A NEK binding motif (F/LXXS/T) is identified in the glycosylation-rich region of PD-L1. NEK2 interacts with PD-L1, phosphorylating the T194/T210 residues and preventing ubiquitin-proteasome pathway-mediated degradation of PD-L1 in ER lumen. NEK2 inhibition thereby sensitizes PD-L1 blockade, synergically enhancing the anti-pancreatic cancer immune response. Together, the present study proposes a promising strategy for improving the effectiveness of pancreatic cancer immunotherapy.
Topics: Adenocarcinoma; Amino Acid Motifs; Amino Acid Sequence; Animals; B7-H1 Antigen; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Immunity; Male; Mice, Nude; Middle Aged; Models, Biological; NIMA-Related Kinases; Pancreatic Neoplasms; Phosphorylation; Phosphoserine; Prognosis; Proteasome Endopeptidase Complex; Protein Binding; Protein Stability; Proteolysis; Ubiquitination; Mice
PubMed: 34315872
DOI: 10.1038/s41467-021-24769-3 -
Molecular Cell Oct 2020Stabilization of stalled replication forks is a prominent mechanism of PARP (Poly(ADP-ribose) Polymerase) inhibitor (PARPi) resistance in BRCA-deficient tumors....
Stabilization of stalled replication forks is a prominent mechanism of PARP (Poly(ADP-ribose) Polymerase) inhibitor (PARPi) resistance in BRCA-deficient tumors. Epigenetic mechanisms of replication fork stability are emerging but remain poorly understood. Here, we report the histone acetyltransferase PCAF (p300/CBP-associated) as a fork-associated protein that promotes fork degradation in BRCA-deficient cells by acetylating H4K8 at stalled replication forks, which recruits MRE11 and EXO1. A H4K8ac binding domain within MRE11/EXO1 is required for their recruitment to stalled forks. Low PCAF levels, which we identify in a subset of BRCA2-deficient tumors, stabilize stalled forks, resulting in PARPi resistance in BRCA-deficient cells. Furthermore, PCAF activity is tightly regulated by ATR (ataxia telangiectasia and Rad3-related), which phosphorylates PCAF on serine 264 (S264) to limit its association and activity at stalled forks. Our results reveal PCAF and histone acetylation as critical regulators of fork stability and PARPi responses in BRCA-deficient cells, which provides key insights into targeting BRCA-deficient tumors and identifying epigenetic modulators of chemotherapeutic responses.
Topics: Acetylation; Amino Acid Sequence; Ataxia Telangiectasia Mutated Proteins; BRCA1 Protein; BRCA2 Protein; Breast Neoplasms; Cell Line, Tumor; DNA Repair Enzymes; DNA Replication; Exodeoxyribonucleases; Female; Gene Expression Regulation, Neoplastic; Histones; Humans; Lysine; MRE11 Homologue Protein; Models, Biological; Mutation; Phosphorylation; Phosphoserine; Poly(ADP-ribose) Polymerase Inhibitors; Protein Binding; p300-CBP Transcription Factors
PubMed: 32966758
DOI: 10.1016/j.molcel.2020.08.018 -
Proceedings of the National Academy of... Jul 2021Although inflammation is critical for the clearance of pathogens, uncontrolled inflammation also contributes to the development of multiple diseases such as cancer and...
Although inflammation is critical for the clearance of pathogens, uncontrolled inflammation also contributes to the development of multiple diseases such as cancer and sepsis. Since NF-κB-mediated transactivation in the nucleus is pivotal downstream of various stimuli to induce inflammation, searching the nuclear-localized targets specifically regulating NF-κB activation will provide important therapeutic application. Here, we have identified that homeodomain-interacting protein kinase 2 (HIPK2), a nuclear serine/threonine kinase, increases its expression in inflammatory macrophages. Importantly, HIPK2 deficiency or overexpression could enhance or inhibit inflammatory responses in LPS-stimulated macrophages, respectively. HIPK2-deficient mice were more susceptible to LPS-induced endotoxemia and CLP-induced sepsis. Adoptive transfer of bone marrow cells (BMs) also aggravated AOM/DSS-induced colorectal cancer. Mechanistically, HIPK2 bound and phosphorylated histone deacetylase 3 (HDAC3) at serine 374 to inhibit its enzymatic activity, thus reducing the deacetylation of p65 at lysine 218 to suppress NF-κB activation. Notably, the HDAC3 inhibitors protected wild-type or BMs-reconstituted mice from LPS-induced endotoxemia. Our findings suggest that the HIPK2-HDAC3-p65 module in macrophages restrains excessive inflammation, which may represent a new layer of therapeutic mechanism for colitis-associated colorectal cancer and sepsis.
Topics: Acetylation; Animals; Cecum; Colitis; Colorectal Neoplasms; Cytokines; Endotoxemia; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Inflammation; Inflammation Mediators; Ligation; Lipopolysaccharides; Lysine; Macrophages; Mice; NF-kappa B; Phosphorylation; Phosphoserine; Protein Serine-Threonine Kinases; Punctures; Sepsis; Toll-Like Receptor 4; Toll-Like Receptor 9; Transcription Factor RelA; Up-Regulation
PubMed: 34244427
DOI: 10.1073/pnas.2021798118