bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2022–03–06
27 papers selected by
Ralitsa Radostinova Madsen, University College London
  1. Phenotypic and molecular characterization of five patients with PIK3CA-related overgrowth spectrum (PROS).
  2. Fyn and TOM1L1 are recruited to clathrin-coated pits and regulate Akt signaling.
  3. Distinct metabolic profiles associated with autism spectrum disorder versus cancer in individuals with germline PTEN mutations.
  4. Insulin facilitates corneal wound healing in the diabetic environment through the RTK-PI3K/Akt/mTOR axis in vitro.
  5. In vitro anticancer effects of alpelisib against PIK3CA‑mutated canine hemangiosarcoma cell lines.
  6. Inhibition of MEK-ERK signaling reduces seizures in two mouse models of tuberous sclerosis complex.
  7. Brachial plexus lipomatosis with perineurial pseudoonion bulb formation: Result of a mosaic PIK3CA mutation in the para-axial mesoderm state.
  8. An omic and multidimensional spatial atlas from serial biopsies of an evolving metastatic breast cancer.
  9. Antispacer peptide nucleic acids for sequence-specific CRISPR-Cas9 modulation.
  10. Autocrine signaling can explain the emergence of Allee effects in cancer cell populations.
  11. Premature aging in mice with error-prone protein synthesis.
  12. Activation of Rictor/mTORC2 signaling acts as a pivotal strategy to protect against sensorineural hearing loss.
  13. Cancer proteogenomics: current impact and future prospects.
  14. Silent mutations reveal therapeutic vulnerability in RAS Q61 cancers.
  15. Signalling dynamics, cell decisions, and homeostatic control in health and disease.
  16. Mutation-specific reporter for optimization and enrichment of prime editing.
  17. Reconstruction and analysis of a large-scale binary Ras-effector signaling network.
  18. Insulin and cancer: a tangled web.
  19. Molecular hallmarks of heterochronic parabiosis at single-cell resolution.
  20. Ultra-high sensitivity mass spectrometry quantifies single-cell proteome changes upon perturbation.
  21. O-GlcNAcylation regulates epidermal growth factor receptor intracellular trafficking and signaling.
  22. Receptor tyrosine kinases regulate signal transduction through a liquid-liquid phase separated state.
  23. Synthetic mammalian signaling circuits for robust cell population control.
  24. Insulin-induced palmitoylation regulates the Cardiac Na+/Ca2+ exchanger NCX1.
  25. Metabolic incorporation of electron-rich ribonucleosides enhances APEX-seq for profiling spatially restricted nascent transcriptome.
  26. Exploiting senescence for the treatment of cancer.
  27. Louder for longer: Myc amplifies gene expression by extended transcriptional bursting.
  1. Am J Med Genet A. 2022 Mar 02.
    Phenotypic and molecular characterization of five patients with PIK3CA-related overgrowth spectrum (PROS).
    Ezgi Gökpınar İli, Elifcan Taşdelen, Ceren Damla Durmaz, Şule Altıner, Timur Tuncalı, Victor Martinez-Glez, Halil Gürhan Karabulut, Seçil Vural, Serdar Ceylaner, Mustafa Oğuz Acar, Hatice Ilgın Ruhi.
      Somatic and germline PI3K-AKT-mTOR pathway pathogenic variants are involved in several segmental overgrowth phenotypes such as the PIK3CA-related overgrowth spectrum (PROS), Proteus syndrome, and PTEN hamartoma tumor syndrome. In this study, we describe five patients with PROS. We identified by high-throughput sequencing four different somatic PIK3CA pathogenic variants in five individuals. The Glu726Lys variant, which was previously reported in megalencephaly-capillary malformation-polymicrogyria (MCAP) syndrome, was identified in two patients with unclassified PROS. The Cys420Arg substitution, which was previously reported in CLOVES, was found in a patient with fibroadipose hyperplasia. Additionally, relatively rare pathogenic variants, His1047Tyr and Tyr1021Cys, were detected in two patients with MCAP. Therefore, we suggest performing deep sequencing of PIK3CA in all patients with suspected PROS, instead of targeted polymerase chain reaction for hotspot pathogenic variants.
    Keywords:  PIK3CA; PROS; overgrowth spectrum; somatic mosaicism
    DOI:  https://doi.org/10.1002/ajmg.a.62709
  2. J Cell Biol. 2022 Apr 04. pii: e201808181. [Epub ahead of print]221(4):
    Fyn and TOM1L1 are recruited to clathrin-coated pits and regulate Akt signaling.
    Rebecca Cabral-Dias, Stefanie Lucarelli, Karolina Zak, Sadia Rahmani, Gurjeet Judge, John Abousawan, Laura F DiGiovanni, Dafne Vural, Karen E Anderson, Michael G Sugiyama, Gizem Genc, Wanjin Hong, Roberto J Botelho, Gregory D Fairn, Peter K Kim, Costin N Antonescu.
      The epidermal growth factor (EGF) receptor (EGFR) controls many aspects of cell physiology. EGF binding to EGFR elicits the membrane recruitment and activation of phosphatidylinositol-3-kinase, leading to Akt phosphorylation and activation. Concomitantly, EGFR is recruited to clathrin-coated pits (CCPs), eventually leading to receptor endocytosis. Previous work uncovered that clathrin, but not receptor endocytosis, is required for EGF-stimulated Akt activation, and that some EGFR signals are enriched in CCPs. Here, we examine how CCPs control EGFR signaling. The signaling adaptor TOM1L1 and the Src-family kinase Fyn are enriched within a subset of CCPs with unique lifetimes and protein composition. Perturbation of TOM1L1 or Fyn impairs EGF-stimulated phosphorylation of Akt2 but not Akt1. EGF stimulation also triggered the TOM1L1- and Fyn-dependent recruitment of the phosphoinositide 5-phosphatase SHIP2 to CCPs. Thus, the recruitment of TOM1L1 and Fyn to a subset of CCPs underlies a role for these structures in the support of EGFR signaling leading to Akt activation.
    DOI:  https://doi.org/10.1083/jcb.201808181
  3. NPJ Genom Med. 2022 Mar 03. 7(1): 16
    Distinct metabolic profiles associated with autism spectrum disorder versus cancer in individuals with germline PTEN mutations.
    Lamis Yehia, Ying Ni, Tammy Sadler, Thomas W Frazier, Charis Eng.
      PTEN hamartoma tumor syndrome (PHTS), caused by germline PTEN mutations, has been associated with organ-specific cancers and autism spectrum disorder (ASD) and/or developmental delay (DD). Predicting precise clinical phenotypes in any one PHTS individual remains impossible. We conducted an untargeted metabolomics study on an age- and sex-matched series of PHTS individuals with ASD/DD, cancer, or both phenotypes. Using agnostic metabolomic-analyses from patient-derived lymphoblastoid cells and their spent media, we found 52 differentially abundant individual metabolites, 69 cell/media metabolite ratios, and 327 pair-wise metabotype (shared metabolic phenotype) ratios clearly distinguishing PHTS individuals based on phenotype. Network analysis based on significant metabolites pointed to hubs converging on PTEN-related insulin, MAPK, AMPK, and mTOR signaling cascades. Internal cross-validation of significant metabolites showed optimal overall accuracy in distinguishing PHTS individuals with ASD/DD versus those with cancer. Such metabolomic markers may enable more accurate risk predictions and prevention in individual PHTS patients at highest risk.
    DOI:  https://doi.org/10.1038/s41525-022-00289-x
  4. Mol Cell Endocrinol. 2022 Feb 26. pii: S0303-7207(22)00058-2. [Epub ahead of print]548 111611
    Insulin facilitates corneal wound healing in the diabetic environment through the RTK-PI3K/Akt/mTOR axis in vitro.
    C Peterson, H L Chandler.
      Diabetic patients can develop degenerative corneal changes, termed diabetic keratopathy, during the course of their disease. Topical insulin has been shown to reduce corneal wound area and restore sensitivity in diabetic rats, and both the insulin receptor (IR) and insulin-like growth factor 1 receptor (IGF-1R) stimulate cell signaling of the PI3K-Akt pathway. The purpose of this study was to assess a mechanism by which improved wound healing occurs by characterizing expression within the PI3K-Akt pathway in corneal epithelial and stromal cells. In vitro scratch tests were used to evaluate wound healing outcomes under variable glucose conditions in the presence or absence of insulin. Protein expression of intracellular kinases in the PI3K pathway, stromal cell markers, and GLUT-1 was evaluated by immunoblotting.TGF-β1 expression was evaluated by ELISA. Insulin promoted in vitro wound healing in all cell types. In human corneal epithelial cells, insulin did not induce PI3K-Akt signaling; however, in all other cell types evaluated, insulin increased expression of PI3K-Akt signaling proteins compared to vehicle control. Fibroblasts variably expressed α-SMA under all treatment conditions, with significant increases in α-SMA and TGF-β1 occurring in a dose-dependent manner with glucose concentration. These results indicate that insulin can promote corneal cellular migration and proliferation by inducing Akt signaling. Exogenous insulin therapy may serve as a novel target of therapeutic intervention for diabetic keratopathy.
    Keywords:  Alpha-smooth muscle actin (α-SMA); Corneal wound healing; Diabetes mellitus; Glucose; Insulin; Transforming growth factor-beta (TGF-β)
    DOI:  https://doi.org/10.1016/j.mce.2022.111611
  5. Oncol Rep. 2022 Apr;pii: 84. [Epub ahead of print]47(4):
    In vitro anticancer effects of alpelisib against PIK3CA‑mutated canine hemangiosarcoma cell lines.
    Marika Maeda, Kazuhiko Ochiai, Masaki Michishita, Masami Morimatsu, Hiroki Sakai, Nayuta Kinoshita, Motoharu Sakaue, Eri Onozawa, Daigo Azakami, Masami Yamamoto, Katsumi Ishioka, Takuya Sadahira, Masami Watanabe, Yoshikazu Tanaka.
      Hemangiosarcoma (HSA) is a malignant neoplasm that occurs in humans and canines with a poor prognosis owing to metastatic spread, despite effective treatment. The frequency of spontaneous HSA development is higher in canines than in humans. Therefore, canine HSA is a useful model of intractable human disease, which requires early detection and an effective therapeutic strategy. A high frequency of the p110α phosphatidylinositol‑4,5‑bisphosphate 3‑kinase catalytic subunit alpha (PIK3CA) mutations is detected in a comprehensive genome‑wide analysis of canine cases of HSA. The present cloned the full‑length cDNA of canine PIK3CA and identified a mutation in codon 1047 from canine cases of HSA and cell lines that were established from these. The enforced expression of the 1047th histidine residue (H1047)R or L mutants of canine PIK3CA in HeLa cells enhanced epidermal growth factor receptor (EGFR) signaling via Akt phosphorylation. PIK3CA mutant canine HSA cell lines exhibited the hyperphosphorylation of Akt upon EGF stimulation as well. Alpelisib, a molecular targeted drug against PIK3CA activating mutations, exerted a significant antitumor effect in canine PIK3CA‑mutated HSA cell lines. By contrast, it had no significant effect on canine mammary gland tumor cell lines harboring PIK3CA mutations. On the whole, the findings of the present study suggest that alpelisib may be highly effective against PIK3CA mutations that occur frequently in canine HSA.
    Keywords:  5‑bisphosphate 3‑kinase catalytic subunit alpha; Akt; alpelisib; canine; hemangiosarcoma; mutation; p110α phosphatidylinositol‑4
    DOI:  https://doi.org/10.3892/or.2022.8295
  6. Epilepsy Res. 2022 Feb 18. pii: S0920-1211(22)00041-9. [Epub ahead of print]181 106890
    Inhibition of MEK-ERK signaling reduces seizures in two mouse models of tuberous sclerosis complex.
    Lena H Nguyen, Steven C Leiser, Dekun Song, Daniela Brunner, Steven L Roberds, Michael Wong, Angelique Bordey.
      Tuberous sclerosis complex (TSC) is a monogenic disorder characterized by hyperactivation of the mTOR signaling pathway and developmental brain malformations leading to intractable epilepsy. Although treatment with the recently approved mTOR inhibitor, everolimus, results in clinically relevant seizure suppression in up to 40% of TSC patients, seizures remain uncontrolled in a large number of cases, underscoring the need to identify novel treatment targets. The MEK-ERK signaling pathway has been found to be aberrantly activated in TSC and inhibition of MEK-ERK activity independently of mTOR rescued neuronal dendrite overgrowth in mice modeling TSC neuropathology. Here, we evaluated the efficacy of MEK-ERK inhibition on seizures in two mouse models of TSC. We found that treatment with the MEK inhibitor PD0325901 (mirdametinib) significantly reduced seizure activity in both TSC mouse models. These findings support inhibiting MEK-ERK activity as a potential alternative strategy to treat seizures in TSC.
    Keywords:  Epilepsy; MAPK; MEK inhibitor; MEK-ERK signaling; Seizures; Tuberous sclerosis complex
    DOI:  https://doi.org/10.1016/j.eplepsyres.2022.106890
  7. Brain Pathol. 2022 Feb 27. e13057
    Brachial plexus lipomatosis with perineurial pseudoonion bulb formation: Result of a mosaic PIK3CA mutation in the para-axial mesoderm state.
    Blake A Ebner, Kathryn L Eschbacher, Megan M Jack, Milosevic Dragana, Robert J Spinner, Caterina Giannini.
      
    Keywords:  PIK3CA-related overgrowth spectrum; brachial plexus; lipomatosis of nerve; perineurial cell pseudo-onion bulb; segmental overgrowth
    DOI:  https://doi.org/10.1111/bpa.13057
  8. Cell Rep Med. 2022 Feb 15. 3(2): 100525
    An omic and multidimensional spatial atlas from serial biopsies of an evolving metastatic breast cancer.
    Brett E Johnson, Allison L Creason, Jayne M Stommel, Jamie M Keck, Swapnil Parmar, Courtney B Betts, Aurora Blucher, Christopher Boniface, Elmar Bucher, Erik Burlingame, Todd Camp, Koei Chin, Jennifer Eng, Joseph Estabrook, Heidi S Feiler, Michael B Heskett, Zhi Hu, Annette Kolodzie, Ben L Kong, Marilyne Labrie, Jinho Lee, Patrick Leyshock, Souraya Mitri, Janice Patterson, Jessica L Riesterer, Shamilene Sivagnanam, Julia Somers, Damir Sudar, Guillaume Thibault, Benjamin R Weeder, Christina Zheng, Xiaolin Nan, Reid F Thompson, Laura M Heiser, Paul T Spellman, George Thomas, Emek Demir, Young Hwan Chang, Lisa M Coussens, Alexander R Guimaraes, Christopher Corless, Jeremy Goecks, Raymond Bergan, Zahi Mitri, Gordon B Mills, Joe W Gray.
      Mechanisms of therapeutic resistance and vulnerability evolve in metastatic cancers as tumor cells and extrinsic microenvironmental influences change during treatment. To support the development of methods for identifying these mechanisms in individual people, here we present an omic and multidimensional spatial (OMS) atlas generated from four serial biopsies of an individual with metastatic breast cancer during 3.5 years of therapy. This resource links detailed, longitudinal clinical metadata that includes treatment times and doses, anatomic imaging, and blood-based response measurements to clinical and exploratory analyses, which includes comprehensive DNA, RNA, and protein profiles; images of multiplexed immunostaining; and 2- and 3-dimensional scanning electron micrographs. These data report aspects of heterogeneity and evolution of the cancer genome, signaling pathways, immune microenvironment, cellular composition and organization, and ultrastructure. We present illustrative examples of how integrative analyses of these data reveal potential mechanisms of response and resistance and suggest novel therapeutic vulnerabilities.
    Keywords:  human tumor atlas; metastatic breast cancer; personalized medicine; precision oncology
    DOI:  https://doi.org/10.1016/j.xcrm.2022.100525
  9. Nucleic Acids Res. 2022 Mar 03. pii: gkac095. [Epub ahead of print]
    Antispacer peptide nucleic acids for sequence-specific CRISPR-Cas9 modulation.
    Nicholas G Economos, Elias Quijano, Kelly E W Carufe, J Dinithi R Perera, Peter M Glazer.
      Despite the rapid and broad implementation of CRISPR-Cas9-based technologies, convenient tools to modulate dose, timing, and precision remain limited. Building on methods using synthetic peptide nucleic acids (PNAs) to bind RNA with unusually high affinity, we describe guide RNA (gRNA) spacer-targeted, or 'antispacer', PNAs as a tool to modulate Cas9 binding and activity in cells in a sequence-specific manner. We demonstrate that PNAs rapidly and efficiently target complexed gRNA spacer sequences at low doses and without design restriction for sequence-selective Cas9 inhibition. We further show that short PAM-proximal antispacer PNAs achieve potent cleavage inhibition (over 2000-fold reduction) and that PAM-distal PNAs modify gRNA affinity to promote on-target specificity. Finally, we apply antispacer PNAs for temporal regulation of two dCas9-fusion systems. These results present a novel rational approach to nucleoprotein engineering and describe a rapidly implementable antisense platform for CRISPR-Cas9 modulation to improve spatiotemporal versatility and safety across applications.
    DOI:  https://doi.org/10.1093/nar/gkac095
  10. PLoS Comput Biol. 2022 Mar 03. 18(3): e1009844
    Autocrine signaling can explain the emergence of Allee effects in cancer cell populations.
    Philip Gerlee, Philipp M Altrock, Adam Malik, Cecilia Krona, Sven Nelander.
      In many human cancers, the rate of cell growth depends crucially on the size of the tumour cell population. Low, zero, or negative growth at low population densities is known as the Allee effect; this effect has been studied extensively in ecology, but so far lacks a good explanation in the cancer setting. Here, we formulate and analyze an individual-based model of cancer, in which cell division rates are increased by the local concentration of an autocrine growth factor produced by the cancer cells themselves. We show, analytically and by simulation, that autocrine signaling suffices to cause both strong and weak Allee effects. Whether low cell densities lead to negative (strong effect) or reduced (weak effect) growth rate depends directly on the ratio of cell death to proliferation, and indirectly on cellular dispersal. Our model is consistent with experimental observations from three patient-derived brain tumor cell lines grown at different densities. We propose that further studying and quantifying population-wide feedback, impacting cell growth, will be central for advancing our understanding of cancer dynamics and treatment, potentially exploiting Allee effects for therapy.
    DOI:  https://doi.org/10.1371/journal.pcbi.1009844
  11. Sci Adv. 2022 Mar 04. 8(9): eabl9051
    Premature aging in mice with error-prone protein synthesis.
    Dimitri Shcherbakov, Martina Nigri, Rashid Akbergenov, Margarita Brilkova, Matilde Mantovani, Patricia Isnard Petit, Amandine Grimm, Agnieszka A Karol, Youjin Teo, Adrián Cortés Sanchón, Yadhu Kumar, Anne Eckert, Kader Thiam, Petra Seebeck, David P Wolfer, Erik C Böttger.
      The main source of error in gene expression is messenger RNA decoding by the ribosome. Translational accuracy has been suggested on a purely correlative basis to positively coincide with maximum possible life span among different rodent species, but causal evidence that translation errors accelerate aging in vivo and limit life span is lacking. We have now addressed this question experimentally by creating heterozygous knock-in mice that express the ribosomal ambiguity mutation RPS9 D95N, resulting in genome-wide error-prone translation. Here, we show that Rps9 D95N knock-in mice exhibit reduced life span and a premature onset of numerous aging-related phenotypes, such as reduced weight, chest deformation, hunchback posture, poor fur condition, and urinary syndrome, together with lymphopenia, increased levels of reactive oxygen species-inflicted damage, accelerated age-related changes in DNA methylation, and telomere attrition. Our results provide an experimental link between translational accuracy, life span, and aging-related phenotypes in mammals.
    DOI:  https://doi.org/10.1126/sciadv.abl9051
  12. Proc Natl Acad Sci U S A. 2022 Mar 08. 119(10): e2107357119
    Activation of Rictor/mTORC2 signaling acts as a pivotal strategy to protect against sensorineural hearing loss.
    Xiaolong Fu, Peipei Li, Linqing Zhang, Yuning Song, Yachun An, Aizhen Zhang, Wenwen Liu, Chao Ye, Yuan Zhang, Rongyu Yue, Xiaoyang Sun, Renjie Chai, Haibo Wang, Jiangang Gao.
      Significance The mechanistic target of rapamycin (mTOR) plays a central role in growth, metabolism, and aging. It is assembled into two multiprotein complexes, namely, mTORC1 and mTORC2. We previously demonstrated the efficacy of sirolimus in ARHL in mice by decreasing mTORC1. However, the aspect of mTORC2 regulation in the cochlea is poorly characterized. Herein, based on pharmacological and genetic interventions, we found that a high dose of sirolimus resulted in severe hearing loss by reducing the mTORC2/AKT signaling pathway in the cochlea. Furthermore, selective activation of mTORC2 could protect against hearing loss induced by acoustic trauma and cisplatin-induced ototoxicity. Hence, the therapeutic activation of mTORC2 in conjunction with decreasing mTORC1 might represent a promising and effective strategy in preventing hearing loss.
    Keywords:  hair cells; hearing; mTORC2
    DOI:  https://doi.org/10.1073/pnas.2107357119
  13. Nat Rev Cancer. 2022 Mar 02.
    Cancer proteogenomics: current impact and future prospects.
    D R Mani, Karsten Krug, Bing Zhang, Shankha Satpathy, Karl R Clauser, Li Ding, Matthew Ellis, Michael A Gillette, Steven A Carr.
      Genomic analyses in cancer have been enormously impactful, leading to the identification of driver mutations and development of targeted therapies. But the functions of the vast majority of somatic mutations and copy number variants in tumours remain unknown, and the causes of resistance to targeted therapies and methods to overcome them are poorly defined. Recent improvements in mass spectrometry-based proteomics now enable direct examination of the consequences of genomic aberrations, providing deep and quantitative characterization of tumour tissues. Integration of proteins and their post-translational modifications with genomic, epigenomic and transcriptomic data constitutes the new field of proteogenomics, and is already leading to new biological and diagnostic knowledge with the potential to improve our understanding of malignant transformation and therapeutic outcomes. In this Review we describe recent developments in proteogenomics and key findings from the proteogenomic analysis of a wide range of cancers. Considerations relevant to the selection and use of samples for proteogenomics and the current technologies used to generate, analyse and integrate proteomic with genomic data are described. Applications of proteogenomics in translational studies and immuno-oncology are rapidly emerging, and the prospect for their full integration into therapeutic trials and clinical care seems bright.
    DOI:  https://doi.org/10.1038/s41568-022-00446-5
  14. Nature. 2022 Mar 02.
    Silent mutations reveal therapeutic vulnerability in RAS Q61 cancers.
    Yoshihisa Kobayashi, Chhayheng Chhoeu, Jiaqi Li, Kristin S Price, Lesli A Kiedrowski, Jamie L Hutchins, Aaron I Hardin, Zihan Wei, Fangxin Hong, Magda Bahcall, Prafulla C Gokhale, Pasi A Jänne.
      RAS family members are the most frequently mutated oncogenes in human cancers. Although KRAS(G12C)-specific inhibitors show clinical activity in patients with cancer1-3, there are no direct inhibitors of NRAS, HRAS or non-G12C KRAS variants. Here we uncover the requirement of the silent KRASG60G mutation for cells to produce a functional KRAS(Q61K). In the absence of this G60G mutation in KRASQ61K, a cryptic splice donor site is formed, promoting alternative splicing and premature protein termination. A G60G silent mutation eliminates the splice donor site, yielding a functional KRAS(Q61K) variant. We detected a concordance of KRASQ61K and a G60G/A59A silent mutation in three independent pan-cancer cohorts. The region around RAS Q61 is enriched in exonic splicing enhancer (ESE) motifs and we designed mutant-specific oligonucleotides to interfere with ESE-mediated splicing, rendering the RAS(Q61) protein non-functional in a mutant-selective manner. The induction of aberrant splicing by antisense oligonucleotides demonstrated therapeutic effects in vitro and in vivo. By studying the splicing necessary for a functional KRAS(Q61K), we uncover a mutant-selective treatment strategy for RASQ61 cancer and expose a mutant-specific vulnerability, which could potentially be exploited for therapy in other genetic contexts.
    DOI:  https://doi.org/10.1038/s41586-022-04451-4
  15. Curr Opin Cell Biol. 2022 Mar 01. pii: S0955-0674(22)00012-6. [Epub ahead of print]75 102066
    Signalling dynamics, cell decisions, and homeostatic control in health and disease.
    Pablo Oriol Valls, Alessandro Esposito.
      Cell signalling engenders cells with the capability to receive and process information from the intracellular and extracellular environments, trigger and execute biological responses, and communicate with each other. Ultimately, cell signalling is responsible for maintaining homeostasis at the cellular, tissue and systemic level. For this reason, cell signalling is a topic of intense research efforts aimed to elucidate how cells coordinate transitions between states in developing and adult organisms in physiological and pathological conditions. Here, we review current knowledge of how cell signalling operates at multiple spatial and temporal scales, focusing on how single-cell analytical techniques reveal mechanisms underpinning cell-to-cell variability, signalling plasticity, and collective cellular responses.
    DOI:  https://doi.org/10.1016/j.ceb.2022.01.011
  16. Nat Commun. 2022 Mar 01. 13(1): 1028
    Mutation-specific reporter for optimization and enrichment of prime editing.
    I F Schene, I P Joore, J H L Baijens, R Stevelink, G Kok, S Shehata, E F Ilcken, E C M Nieuwenhuis, D P Bolhuis, R C M van Rees, S A Spelier, H P J van der Doef, J M Beekman, R H J Houwen, E E S Nieuwenhuis, S A Fuchs.
      Prime editing is a versatile genome-editing technique that shows great promise for the generation and repair of patient mutations. However, some genomic sites are difficult to edit and optimal design of prime-editing tools remains elusive. Here we present a fluorescent prime editing and enrichment reporter (fluoPEER), which can be tailored to any genomic target site. This system rapidly and faithfully ranks the efficiency of prime edit guide RNAs (pegRNAs) combined with any prime editor variant. We apply fluoPEER to instruct correction of pathogenic variants in patient cells and find that plasmid editing enriches for genomic editing up to 3-fold compared to conventional enrichment strategies. DNA repair and cell cycle-related genes are enriched in the transcriptome of edited cells. Stalling cells in the G1/S boundary increases prime editing efficiency up to 30%. Together, our results show that fluoPEER can be employed for rapid and efficient correction of patient cells, selection of gene-edited cells, and elucidation of cellular mechanisms needed for successful prime editing.
    DOI:  https://doi.org/10.1038/s41467-022-28656-3
  17. Cell Commun Signal. 2022 Mar 04. 20(1): 24
    Reconstruction and analysis of a large-scale binary Ras-effector signaling network.
    Simona Catozzi, Camille Ternet, Alize Gourrege, Kieran Wynne, Giorgio Oliviero, Christina Kiel.
       BACKGROUND: Ras is a key cellular signaling hub that controls numerous cell fates via multiple downstream effector pathways. While pathways downstream of effectors such as Raf, PI3K and RalGDS are extensively described in the literature, how other effectors signal downstream of Ras is often still enigmatic.
    METHODS: A comprehensive and unbiased Ras-effector network was reconstructed downstream of 43 effector proteins (converging onto 12 effector classes) using public pathway and protein-protein interaction (PPI) databases. The output is an oriented graph of pairwise interactions defining a 3-layer signaling network downstream of Ras. The 2290 proteins comprising the network were studied for their implication in signaling crosstalk and feedbacks, their subcellular localizations, and their cellular functions.
    RESULTS: The final Ras-effector network consists of 2290 proteins that are connected via 19,080 binary PPIs, increasingly distributed across the downstream layers, with 441 PPIs in layer 1, 1660 in layer 2, and 16,979 in layer 3. We identified a high level of crosstalk among proteins of the 12 effector classes. A class-specific Ras sub-network was generated in CellDesigner (.xml file) and a functional enrichment analysis thereof shows that 58% of the processes have previously been associated to a respective effector pathway, with the remaining providing insights into novel and unexplored functions of specific effector pathways.
    CONCLUSIONS: Our large-scale and cell general Ras-effector network is a crucial steppingstone towards defining the network boundaries. It constitutes a 'reference interactome' and can be contextualized for specific conditions, e.g. different cell types or biopsy material obtained from cancer patients. Further, it can serve as a basis for elucidating systems properties, such as input-output relationships, crosstalk, and pathway redundancy. Video Abstract.
    Keywords:  Crosstalk; Effectors; Feedbacks; Network hubs; Pathway reconstruction; Ras; Signaling pathways
    DOI:  https://doi.org/10.1186/s12964-022-00823-5
  18. Biochem J. 2022 Mar 18. 479(5): 583-607
    Insulin and cancer: a tangled web.
    Brooks P Leitner, Stephan Siebel, Ngozi D Akingbesote, Xinyi Zhang, Rachel J Perry.
      For a century, since the pioneering work of Otto Warburg, the interwoven relationship between metabolism and cancer has been appreciated. More recently, with obesity rates rising in the U.S. and worldwide, epidemiologic evidence has supported a link between obesity and cancer. A substantial body of work seeks to mechanistically unpack the association between obesity, altered metabolism, and cancer. Without question, these relationships are multifactorial and cannot be distilled to a single obesity- and metabolism-altering hormone, substrate, or factor. However, it is important to understand the hormone-specific associations between metabolism and cancer. Here, we review the links between obesity, metabolic dysregulation, insulin, and cancer, with an emphasis on current investigational metabolic adjuncts to standard-of-care cancer treatment.
    Keywords:  cancer metabolism; diabetes; immunometabolism
    DOI:  https://doi.org/10.1042/BCJ20210134
  19. Nature. 2022 Mar 02.
    Molecular hallmarks of heterochronic parabiosis at single-cell resolution.
    Tabula Muris Consortium
      The ability to slow or reverse biological ageing would have major implications for mitigating disease risk and maintaining vitality1. Although an increasing number of interventions show promise for rejuvenation2, their effectiveness on disparate cell types across the body and the molecular pathways susceptible to rejuvenation remain largely unexplored. Here we performed single-cell RNA sequencing on 20 organs to reveal cell-type-specific responses to young and aged blood in heterochronic parabiosis. Adipose mesenchymal stromal cells, haematopoietic stem cells and hepatocytes are among those cell types that are especially responsive. On the pathway level, young blood invokes new gene sets in addition to reversing established ageing patterns, with the global rescue of genes encoding electron transport chain subunits pinpointing a prominent role of mitochondrial function in parabiosis-mediated rejuvenation. We observed an almost universal loss of gene expression with age that is largely mimicked by parabiosis: aged blood reduces global gene expression, and young blood restores it in select cell types. Together, these data lay the groundwork for a systemic understanding of the interplay between blood-borne factors and cellular integrity.
    DOI:  https://doi.org/10.1038/s41586-022-04461-2
  20. Mol Syst Biol. 2022 Mar;18(3): e10798
    Ultra-high sensitivity mass spectrometry quantifies single-cell proteome changes upon perturbation.
    Andreas-David Brunner, Marvin Thielert, Catherine Vasilopoulou, Constantin Ammar, Fabian Coscia, Andreas Mund, Ole B Hoerning, Nicolai Bache, Amalia Apalategui, Markus Lubeck, Sabrina Richter, David S Fischer, Oliver Raether, Melvin A Park, Florian Meier, Fabian J Theis, Matthias Mann.
      Single-cell technologies are revolutionizing biology but are today mainly limited to imaging and deep sequencing. However, proteins are the main drivers of cellular function and in-depth characterization of individual cells by mass spectrometry (MS)-based proteomics would thus be highly valuable and complementary. Here, we develop a robust workflow combining miniaturized sample preparation, very low flow-rate chromatography, and a novel trapped ion mobility mass spectrometer, resulting in a more than 10-fold improved sensitivity. We precisely and robustly quantify proteomes and their changes in single, FACS-isolated cells. Arresting cells at defined stages of the cell cycle by drug treatment retrieves expected key regulators. Furthermore, it highlights potential novel ones and allows cell phase prediction. Comparing the variability in more than 430 single-cell proteomes to transcriptome data revealed a stable-core proteome despite perturbation, while the transcriptome appears stochastic. Our technology can readily be applied to ultra-high sensitivity analyses of tissue material, posttranslational modifications, and small molecule studies from small cell counts to gain unprecedented insights into cellular heterogeneity in health and disease.
    Keywords:  drug perturbation; low-flow LC-MS; proteomics at single-cell resolution; single-cell heterogeneity; systems biology
    DOI:  https://doi.org/10.15252/msb.202110798
  21. Proc Natl Acad Sci U S A. 2022 Mar 08. 119(10): e2107453119
    O-GlcNAcylation regulates epidermal growth factor receptor intracellular trafficking and signaling.
    Liming Wu, Yaxian Cheng, Didi Geng, Zhiya Fan, Bingyi Lin, Qiang Zhu, Jingchao Li, Weijie Qin, Wen Yi.
      SignificanceEpidermal growth factor receptor (EGFR) is one of the most important membrane receptors that transduce growth signals into cells to sustain cell growth, proliferation, and survival. EGFR signal termination is initiated by EGFR internalization, followed by trafficking through endosomes, and degradation in lysosomes. How this process is regulated is still poorly understood. Here, we show that hepatocyte growth factor regulated tyrosine kinase substrate (HGS), a key protein in the EGFR trafficking pathway, is dynamically modified by a single sugar N-acetylglucosamine. This modification inhibits EGFR trafficking from endosomes to lysosomes, leading to the accumulation of EGFR and prolonged signaling. This study provides an important insight into diseases with aberrant growth factor signaling, such as cancer, obesity, and diabetes.
    Keywords:  EGFR; O-GlcNAcylation; endosomal sorting; membrane receptors
    DOI:  https://doi.org/10.1073/pnas.2107453119
  22. Mol Cell. 2022 Feb 19. pii: S1097-2765(22)00110-1. [Epub ahead of print]
    Receptor tyrosine kinases regulate signal transduction through a liquid-liquid phase separated state.
    Chi-Chuan Lin, Kin Man Suen, Polly-Anne Jeffrey, Lukasz Wieteska, Jessica A Lidster, Peng Bao, Alistair P Curd, Amy Stainthorp, Caroline Seiler, Hans Koss, Eric Miska, Zamal Ahmed, Stephen D Evans, Carmen Molina-París, John E Ladbury.
      The recruitment of signaling proteins into activated receptor tyrosine kinases (RTKs) to produce rapid, high-fidelity downstream response is exposed to the ambiguity of random diffusion to the target site. Liquid-liquid phase separation (LLPS) overcomes this by providing elevated, localized concentrations of the required proteins while impeding competitor ligands. Here, we show a subset of phosphorylation-dependent RTK-mediated LLPS states. We then investigate the formation of phase-separated droplets comprising a ternary complex including the RTK, (FGFR2); the phosphatase, SHP2; and the phospholipase, PLCγ1, which assembles in response to receptor phosphorylation. SHP2 and activated PLCγ1 interact through their tandem SH2 domains via a previously undescribed interface. The complex of FGFR2 and SHP2 combines kinase and phosphatase activities to control the phosphorylation state of the assembly while providing a scaffold for active PLCγ1 to facilitate access to its plasma membrane substrate. Thus, LLPS modulates RTK signaling, with potential consequences for therapeutic intervention.
    Keywords:  FGFR2; Liquid-liquid phase separation (LLPS); Plcγ1; Receptor tyrosine kinases (RTKs); Shp2; kinase activity; phosphatase activity; phospholipase activity
    DOI:  https://doi.org/10.1016/j.molcel.2022.02.005
  23. Cell. 2022 Feb 25. pii: S0092-8674(22)00137-4. [Epub ahead of print]
    Synthetic mammalian signaling circuits for robust cell population control.
    Yitong Ma, Mark W Budde, Michaëlle N Mayalu, Junqin Zhu, Andrew C Lu, Richard M Murray, Michael B Elowitz.
      In multicellular organisms, cells actively sense and control their own population density. Synthetic mammalian quorum-sensing circuits could provide insight into principles of population control and extend cell therapies. However, a key challenge is reducing their inherent sensitivity to "cheater" mutations that evade control. Here, we repurposed the plant hormone auxin to enable orthogonal mammalian cell-cell communication and quorum sensing. We designed a paradoxical population control circuit, termed "Paradaux," in which auxin stimulates and inhibits net cell growth at different concentrations. This circuit limited population size over extended timescales of up to 42 days of continuous culture. By contrast, when operating in a non-paradoxical regime, population control became more susceptible to mutational escape. These results establish auxin as a versatile "private" communication system and demonstrate that paradoxical circuit architectures can provide robust population control.
    Keywords:  auxin; cell population control; mammalian synthetic biology; paradoxical control; quorum sensing; synthetic circuits; synthetic signaling
    DOI:  https://doi.org/10.1016/j.cell.2022.01.026
  24. Cell Calcium. 2022 Feb 23. pii: S0143-4160(22)00042-2. [Epub ahead of print]104 102567
    Insulin-induced palmitoylation regulates the Cardiac Na+/Ca2+ exchanger NCX1.
    Caglar Gök, Alan D Robertson, William Fuller.
      The cardiac Na+/Ca2+ Exchanger (NCX1) controls Ca2+ extrusion from the cytosol by mediating bidirectional exchange of Na+ for Ca2+, and therefore controls cardiac relaxation. Insulin regulates Ca2+ handling in cardiac tissue through NCX1, however how insulin changes NCX1 activity is poorly understood. Palmitoylation is the only post-translational modification identified to alter NCX1 activity. Here we show that insulin triggers local structural re-arrangements within existing NCX1 dimers by inducing their palmitoylation, thus tunes NCX1 inactivation through a zDHHC5-dependent mechanism in multiple cell types. By activating fatty acid and fatty acyl CoA synthesis insulin promotes palmitoylation of the zDHHC5 active site, which leads to enhanced NCX1 palmitoylation. Our findings represent a new mechanism to regulate the palmitoylation of numerous zDHHC5 substrates.
    Keywords:  Acylation; Cardiac function; Diabetes; Fatty acylCoA; zDHHC-PAT
    DOI:  https://doi.org/10.1016/j.ceca.2022.102567
  25. Cell Chem Biol. 2022 Feb 28. pii: S2451-9456(22)00086-1. [Epub ahead of print]
    Metabolic incorporation of electron-rich ribonucleosides enhances APEX-seq for profiling spatially restricted nascent transcriptome.
    Ran Li, Zhongyu Zou, Wentao Wang, Peng Zou.
      The spatial arrangement of newly synthesized transcriptome in eukaryotic cells underlies various biological processes including cell proliferation and differentiation. In this study, we combine metabolic incorporation of electron-rich ribonucleosides (e.g., 6-thioguanosine and 4-thiouridine) with a peroxidase-mediated proximity-dependent RNA labeling technique (APEX-seq) to develop a sensitive method, termed MERR APEX-seq, for selectively profiling newly transcribed RNAs at specific subcellular locations in live cells. We demonstrate that MERR APEX-seq is 20-fold more efficient than APEX-seq and offers both high spatial specificity and high coverage in mitochondrial matrix. At the ER membrane, 91% of the transcripts captured by MERR APEX-seq encode for secretory pathway proteins, thus demonstrating the high spatial specificity of MERR APEX-seq in open subcellular compartments. Application of MERR APEX-seq to the nuclear lamina of human cells reveals a local transcriptome of 1,012 RNAs, many of which encode for nuclear proteins involved in histone modification, chromosomal structure maintenance, and RNA processing.
    Keywords:  APEX-seq; RNA labeling; nascent transcriptome; nuclear lamina
    DOI:  https://doi.org/10.1016/j.chembiol.2022.02.005
  26. Nat Rev Cancer. 2022 Mar 03.
    Exploiting senescence for the treatment of cancer.
    Liqin Wang, Lina Lankhorst, René Bernards.
      Senescence is a cellular response to a variety of stress signals that is characterized by a stable withdrawal from the cell cycle and major changes in cell morphology and physiology. While most research on senescence has been performed on non-cancer cells, it is evident that cancer cells can also mount a senescence response. In this Review, we discuss how senescence can be induced in cancer cells. We describe the distinctive features of senescent cancer cells and how these changes in cellular physiology might be exploited for the selective eradication of these cells (senolysis). We discuss activation of the host immune system as a particularly attractive way to clear senescent cancer cells. Finally, we consider the challenges and opportunities provided by a 'one-two punch' sequential treatment of cancer with pro-senescence therapy followed by senolytic therapy.
    DOI:  https://doi.org/10.1038/s41568-022-00450-9
  27. Cell Rep. 2022 Mar 01. pii: S2211-1247(22)00203-0. [Epub ahead of print]38(9): 110470
    Louder for longer: Myc amplifies gene expression by extended transcriptional bursting.
    Dan Lu, Ashwini Jambhekar, Galit Lahav.
      Transcription is a complex, dynamic process. Using live single-cell measurements, Patange et al. show, in a recent issue of Cell Reports, that elevated levels of the transcription factor MYC enhance target gene RNA production by increasing the duration but not frequency of transcriptional bursts.
    DOI:  https://doi.org/10.1016/j.celrep.2022.110470
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