bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2022–05–29
24 papers selected by
Ralitsa Radostinova Madsen, University College London
  1. Pulmonary Vein Stenosis Associated with Germline PIK3CA Mutation.
  2. A human breast atlas integrating single-cell proteomics and transcriptomics.
  3. Spatiotemporal feedforward between PKM2 tetramers and mTORC1 prompts mTORC1 activation.
  4. Developing dietary interventions as therapy for cancer.
  5. Signal requirement for cortical potential of transplantable human neuroepithelial stem cells.
  6. Putting in the Erk: Growth factor signaling and mesoderm morphogenesis.
  7. Mining cell-cell signaling in single-cell transcriptomics atlases.
  8. PIK3CA mutations-mediated downregulation of circLHFPL2 inhibits colorectal cancer progression via upregulating PTEN.
  9. PI(18:1/18:1) is a SCD1-derived lipokine that limits stress signaling.
  10. The impact of phosphorylated PTEN at threonine 366 on cortical connectivity and behaviour.
  11. Fine-Tuning Cardiac Insulin/Insulin-Like Growth Factor 1 Receptor Signaling to Promote Health and Longevity.
  12. Inferring Potential Cancer Driving Synonymous Variants.
  13. Redefining breast cancer subtypes to guide treatment prioritization and maximize response: Predictive biomarkers across 10 cancer therapies.
  14. O-GlcNAc modification of leucyl-tRNA synthetase 1 integrates leucine and glucose availability to regulate mTORC1 and the metabolic fate of leucine.
  15. Differential impact of lipid raft depletion on platelet-derived growth factor (PDGF)-induced ERK1/2 MAP-kinase, SRC and AKT signaling.
  16. Assessing Cell Competition in Human Pluripotent Stem Cell (hPSC) Cultures.
  17. c-JUN inhibits mTORC2 and glucose uptake to promote self-renewal and obesity.
  18. Obesity, Diabetes, and Cancer: The Role of the Insulin/IGF Axis; Mechanisms and Clinical Implications.
  19. The heterogeneity of cellular senescence: insights at the single-cell level.
  20. DRP1 levels determine the apoptotic threshold during embryonic differentiation through a mitophagy-dependent mechanism.
  21. Localization of a TORC1-eIF4F translation complex during CD8+ T cell activation drives divergent cell fate.
  22. Pulling back the curtain: The hidden functions of receptor tyrosine kinases in development.
  23. In Vitro Kinase-to-Phosphosite Database (iKiP-DB) Predicts Kinase Activity in Phosphoproteomic Datasets.
  24. Phosphatidylinositol Phosphates Modulate Interactions between the StarD4 Sterol Trafficking Protein and Lipid Membranes.
  1. Children (Basel). 2022 May 05. pii: 671. [Epub ahead of print]9(5):
    Pulmonary Vein Stenosis Associated with Germline PIK3CA Mutation.
    Delphine Yung, Kaitlyn Freeman, Ghayda Mirzaa.
      Pulmonary vein stenosis is a rare and frequently lethal childhood disease. There are few known genetic associations, and the pathophysiology is not well known. Current treatments include surgery, interventional cardiac catheterization, and more recently, medications targeting cell proliferation, which are not uniformly effective. We present a patient with PVS and a PIK3CA mutation, who demonstrated a good response to the targeted inhibitor, alpelisib.
    Keywords:  PIK3CA; alpelisib; pulmonary vein stenosis
    DOI:  https://doi.org/10.3390/children9050671
  2. Dev Cell. 2022 May 17. pii: S1534-5807(22)00331-8. [Epub ahead of print]
    A human breast atlas integrating single-cell proteomics and transcriptomics.
    G Kenneth Gray, Carman Man-Chung Li, Jennifer M Rosenbluth, Laura M Selfors, Nomeda Girnius, Jia-Ren Lin, Ron C J Schackmann, Walter L Goh, Kaitlin Moore, Hana K Shapiro, Shaolin Mei, Kurt D'Andrea, Katherine L Nathanson, Peter K Sorger, Sandro Santagata, Aviv Regev, Judy E Garber, Deborah A Dillon, Joan S Brugge.
      The breast is a dynamic organ whose response to physiological and pathophysiological conditions alters its disease susceptibility, yet the specific effects of these clinical variables on cell state remain poorly annotated. We present a unified, high-resolution breast atlas by integrating single-cell RNA-seq, mass cytometry, and cyclic immunofluorescence, encompassing a myriad of states. We define cell subtypes within the alveolar, hormone-sensing, and basal epithelial lineages, delineating associations of several subtypes with cancer risk factors, including age, parity, and BRCA2 germline mutation. Of particular interest is a subset of alveolar cells termed basal-luminal (BL) cells, which exhibit poor transcriptional lineage fidelity, accumulate with age, and carry a gene signature associated with basal-like breast cancer. We further utilize a medium-depletion approach to identify molecular factors regulating cell-subtype proportion in organoids. Together, these data are a rich resource to elucidate diverse mammary cell states.
    Keywords:  BRCA1; BRCA2; CyTOF mass cytometry; aging; breast cancer; cell state plasticity; mammary biology; multi-omic single-cell atlas; multiplexed tissue staining; organoids; scRNA-Seq
    DOI:  https://doi.org/10.1016/j.devcel.2022.05.003
  3. Phys Biol. 2022 May 25.
    Spatiotemporal feedforward between PKM2 tetramers and mTORC1 prompts mTORC1 activation.
    Yu Xia, ShuMing Wang, Chunbo Song, Ruoyu Luo.
      Most mammalian cells couple glucose availability to anabolic processes via the mTORC1 pathway. However, the mechanism by which fluctuations in glucose availability are rapidly translated into mTORC1 signals remains elusive. Here, we show that cells rapidly respond to changes in glucose availability through the spatial coupling of mTORC1 and tetramers of the key glycolytic enzyme pyruvate kinase M2 (PKM2) on lysosomal surfaces in the late G1/S phases. The lysosomal localization of PKM2 tetramers enables rapid increases in local ATP concentrations around lysosomes to activate mTORC1, while bypassing the need to elevate global ATP levels in the entire cell. In essence, this spatial coupling establishes a feedforward loop to enable mTORC1 to rapidly sense and respond to changes in glucose availability. We further demonstrate that this mechanism ensures robust cell proliferation upon fluctuating glucose availability. Thus, we present mechanistic insights into the rapid response of the mTORC1 pathway to changes in glucose availability. The underlying mechanism may be applicable to the control of other cellular processes.
    Keywords:  Cancer metabolism; PKM2; Spatiotemporal Feedforward; mTORC1
    DOI:  https://doi.org/10.1088/1478-3975/ac7372
  4. Nat Rev Cancer. 2022 May 25.
    Developing dietary interventions as therapy for cancer.
    Samuel R Taylor, John N Falcone, Lewis C Cantley, Marcus D Goncalves.
      Cancer cells acquire distinct metabolic preferences based on their tissue of origin, genetic alterations and degree of interaction with systemic hormones and metabolites. These adaptations support the increased nutrient demand required for increased growth and proliferation. Diet is the major source of nutrients for tumours, yet dietary interventions lack robust evidence and are rarely prescribed by clinicians for the treatment of cancer. Well-controlled diet studies in patients with cancer are rare, and existing studies have been limited by nonspecific enrolment criteria that inappropriately grouped together subjects with disparate tumour and host metabolic profiles. This imprecision may have masked the efficacy of the intervention for appropriate candidates. Here, we review the metabolic alterations and key vulnerabilities that occur across multiple types of cancer. We describe how these vulnerabilities could potentially be targeted using dietary therapies including energy or macronutrient restriction and intermittent fasting regimens. We also discuss recent trials that highlight how dietary strategies may be combined with pharmacological therapies to treat some cancers, potentially ushering a path towards precision nutrition for cancer.
    DOI:  https://doi.org/10.1038/s41568-022-00485-y
  5. Nat Commun. 2022 May 23. 13(1): 2844
    Signal requirement for cortical potential of transplantable human neuroepithelial stem cells.
    Balazs V Varga, Maryam Faiz, Helena Pivonkova, Gabriel Khelifi, Huijuan Yang, Shangbang Gao, Emma Linderoth, Mei Zhen, Ragnhildur Thora Karadottir, Samer M Hussein, Andras Nagy.
      The cerebral cortex develops from dorsal forebrain neuroepithelial progenitor cells. Following the initial expansion of the progenitor cell pool, these cells generate neurons of all the cortical layers and then astrocytes and oligodendrocytes. Yet, the regulatory pathways that control the expansion and maintenance of the progenitor cell pool are currently unknown. Here we define six basic pathway components that regulate proliferation of cortically specified human neuroepithelial stem cells (cNESCs) in vitro without the loss of cerebral cortex developmental potential. We show that activation of FGF and inhibition of BMP and ACTIVIN A signalling are required for long-term cNESC proliferation. We also demonstrate that cNESCs preserve dorsal telencephalon-specific potential when GSK3, AKT and nuclear CATENIN-β1 activity are low. Remarkably, regulation of these six pathway components supports the clonal expansion of cNESCs. Moreover, cNESCs differentiate into lower- and upper-layer cortical neurons in vitro and in vivo. The identification of mechanisms that drive the neuroepithelial stem cell self-renewal and differentiation and preserve this potential in vitro is key to developing regenerative and cell-based therapeutic approaches to treat neurological conditions.
    DOI:  https://doi.org/10.1038/s41467-022-29839-8
  6. Curr Top Dev Biol. 2022 ;pii: S0070-2153(22)00013-8. [Epub ahead of print]149 263-310
    Putting in the Erk: Growth factor signaling and mesoderm morphogenesis.
    Sarah E McFann, Stanislav Y Shvartsman, Jared E Toettcher.
      It has long been known that FGF signaling contributes to mesoderm formation, a germ layer found in triploblasts that is composed of highly migratory cells that give rise to muscles and to the skeletal structures of vertebrates. FGF signaling activates several pathways in the developing mesoderm, including transient activation of the Erk pathway, which triggers mesodermal fate specification through the induction of the gene brachyury and activates morphogenetic programs that allow mesodermal cells to position themselves in the embryo. In this review, we discuss what is known about the generation and interpretation of transient Erk signaling in mesodermal tissues across species. We focus specifically on mechanisms that translate the level and duration of Erk signaling into cell fate and cell movement instructions and discuss strategies for further interrogating the role that Erk signaling dynamics play in mesodermal gastrulation and morphogenesis.
    Keywords:  Differentiation; Erk; Mesoderm; Signaling dynamics
    DOI:  https://doi.org/10.1016/bs.ctdb.2022.02.007
  7. Curr Opin Cell Biol. 2022 May 21. pii: S0955-0674(22)00054-0. [Epub ahead of print]76 102101
    Mining cell-cell signaling in single-cell transcriptomics atlases.
    Mingxi Deng, Ying Wang, Yan Yan.
      Recent advances in single-cell RNA sequencing (scRNA-seq) techniques lead to an explosion of single-cell atlases from diverse biological contexts. The information of cell-cell signaling events, which underlie multicellular organism function, is embedded in these atlases. Here, we review current strategies of mining cell-cell signaling events from single-cell transcriptomics datasets and highlight examples where functions of predicted cell-cell signaling events from single-cell atlases are further pursued to yield new insights into biological processes.
    DOI:  https://doi.org/10.1016/j.ceb.2022.102101
  8. Mol Cancer. 2022 May 26. 21(1): 118
    PIK3CA mutations-mediated downregulation of circLHFPL2 inhibits colorectal cancer progression via upregulating PTEN.
    Xiaodan Chong, Jingde Chen, Nanxin Zheng, Zhuqing Zhou, Yanan Hai, Shiqing Chen, Yu Zhang, Qingzhuo Yu, Shijun Yu, Zhiqin Chen, Wenfang Bao, Ming Quan, Zhe-Sheng Chen, Yangyang Zhan, Yong Gao.
       BACKGROUND: PIK3CA mutation and PTEN suppression lead to tumorigenesis and drug resistance in colorectal cancer (CRC). There is no research on the role of circular RNAs (circRNAs) in regulating PIK3CA mutation and MEK inhibitor resistance in CRC.
    METHODS: The expression of circLHFPL2 in PIK3CA-mutant and wild-type cells and tissues was quantified by RNA-sequencing and qRT-PCR. CCK-8 assay and colony formation assay were used to evaluate cell viability. Annexin V/PI staining was implemented to assess cell apoptosis. Luciferase assay, biotin-coupled microRNA capture, and RIP assay were used to validate the interaction among potential targets. Western blotting and qRT-PCR assays were used to evaluate the expression of involved targets. Xenograft tumor in a nude mouse model was used to explore the role of circRNAs in vivo.
    RESULTS: RNA sequencing defined downregulated expression of circLHFPL2 in both PIK3CAH1047R (HCT116) and PIK3CAE545K (DLD1) cells. CircLHFPL2 was also downregulated in PIK3CA-mutant CRC primary cells and tissues, which was correlated with poor prognosis. CircLHFPL2 was mainly localized in the cytoplasm and its downregulation was attributed to the PI3K/AKT signaling pathway activated by phosphorylating Foxo3a. CircLHFPL2 inhibited PI3KCA-Mut CRC progression both in vitro and in vivo. Furthermore, our work indicated that circLHFPL2 acts as a ceRNA to sponge miR-556-5p and miR-1322 in CRC cells and in turn modulate the expression of PTEN. Importantly, circLHFPL2 was able to overcome PIK3CA-mediated MEK inhibitor resistance in CRC cells.
    CONCLUSIONS: Downregulation of circLHFPL2 sustains the activation of the PI3K/AKT signaling pathway via a positive feedback loop in PIK3CA-mutant CRC. In addition, downregulation of circLHFPL2 leads to MEK inhibitor resistance in CRC. Therefore, targeting circLHFPL2 could be an effective approach for the treatment of CRC patients harboring oncogenic PIK3CA mutations.
    Keywords:  Colorectal cancer; PI3KCA mutation; PTEN; circLHFPL2; miR-1322; miR-556-5p
    DOI:  https://doi.org/10.1186/s12943-022-01531-x
  9. Nat Commun. 2022 May 27. 13(1): 2982
    PI(18:1/18:1) is a SCD1-derived lipokine that limits stress signaling.
    Maria Thürmer, André Gollowitzer, Helmut Pein, Konstantin Neukirch, Elif Gelmez, Lorenz Waltl, Natalie Wielsch, René Winkler, Konstantin Löser, Julia Grander, Madlen Hotze, Sönke Harder, Annika Döding, Martina Meßner, Fabiana Troisi, Maximilian Ardelt, Hartmut Schlüter, Johanna Pachmayr, Óscar Gutiérrez-Gutiérrez, Karl Lenhard Rudolph, Kathrin Thedieck, Ulrike Schulze-Späte, Cristina González-Estévez, Christian Kosan, Aleš Svatoš, Marcel Kwiatkowski, Andreas Koeberle.
      Cytotoxic stress activates stress-activated kinases, initiates adaptive mechanisms, including the unfolded protein response (UPR) and autophagy, and induces programmed cell death. Fatty acid unsaturation, controlled by stearoyl-CoA desaturase (SCD)1, prevents cytotoxic stress but the mechanisms are diffuse. Here, we show that 1,2-dioleoyl-sn-glycero-3-phospho-(1'-myo-inositol) [PI(18:1/18:1)] is a SCD1-derived signaling lipid, which inhibits p38 mitogen-activated protein kinase activation, counteracts UPR, endoplasmic reticulum-associated protein degradation, and apoptosis, regulates autophagy, and maintains cell morphology and proliferation. SCD1 expression and the cellular PI(18:1/18:1) proportion decrease during the onset of cell death, thereby repressing protein phosphatase 2 A and enhancing stress signaling. This counter-regulation applies to mechanistically diverse death-inducing conditions and is found in multiple human and mouse cell lines and tissues of Scd1-defective mice. PI(18:1/18:1) ratios reflect stress tolerance in tumorigenesis, chemoresistance, infection, high-fat diet, and immune aging. Together, PI(18:1/18:1) is a lipokine that links fatty acid unsaturation with stress responses, and its depletion evokes stress signaling.
    DOI:  https://doi.org/10.1038/s41467-022-30374-9
  10. Brain. 2022 May 23. pii: awac188. [Epub ahead of print]
    The impact of phosphorylated PTEN at threonine 366 on cortical connectivity and behaviour.
    Julia M T Ledderose, Jorge A Benitez, Amanda J Roberts, Rachel Reed, Willem Bintig, Matthew E Larkum, Robert N S Sachdev, Frank Furnari, Britta J Eickholt.
      The lipid phosphatase PTEN (phosphatase and tensin homologue on chromosome 10) is a key tumour suppressor gene and an important regulator of neuronal signalling. PTEN mutations have been identified in patients with autism spectrum disorders, characterized by macrocephaly, impaired social interactions and communication, repetitive behaviour, intellectual disability, and epilepsy. PTEN enzymatic activity is regulated by a cluster of phosphorylation sites at the C-terminus of the protein. Here, we focussed on the role of PTEN T366 phosphorylation and generated a knock-in mouse line in which Pten T366 was substituted with alanine (PtenT366A/T366A). We identify that phosphorylation of PTEN at T366 controls neuron size and connectivity of brain circuits involved in sensory processing. We show in behavioural tests that PtenT366/T366A mice exhibit cognitive deficits and selective sensory impairments, with significant differences in male individuals. We identify restricted cellular overgrowth of cortical neurons in PtenT366A/T366A brains, linked to increases in both dendritic arborization and soma size. In a combinatorial approach of anterograde and retrograde monosynaptic tracing using rabies virus, we characterize differences in connectivity to the primary somatosensory cortex of PtenT366A/T366A brains, with imbalances in long-range cortico-cortical input to neurons. We conclude that phosphorylation of PTEN at T366 controls neuron size and connectivity of brain circuits involved in sensory processing and propose that PTEN T366 signalling may account for a subset of autism-related functions of PTEN.
    Keywords:  PTEN; cognitive behaviour; cortical connectivity; developmental disorders; neuronal morphology
    DOI:  https://doi.org/10.1093/brain/awac188
  11. Circulation. 2022 May 26. 101161CIRCULATIONAHA122059863
    Fine-Tuning Cardiac Insulin/Insulin-Like Growth Factor 1 Receptor Signaling to Promote Health and Longevity.
    Mahmoud Abdellatif, Viktoria Trummer-Herbst, Alexander Martin Heberle, Alina Humnig, Tobias Pendl, Sylvère Durand, Giulia Cerrato, Sebastian J Hofer, Moydul Islam, Julia Voglhuber, José Miguel Ramos Pittol, Oliver Kepp, Gerald Hoefler, Albrecht Schmidt, Peter P Rainer, Daniel Scherr, Dirk von Lewinski, Egbert Bisping, Julie R McMullen, Abhinav Diwan, Tobias Eisenberg, Frank Madeo, Kathrin Thedieck, Guido Kroemer, Simon Sedej.
       BACKGROUND: The insulin/insulin-like growth factor 1 (IGF1) pathway is a key regulator of cellular metabolism and aging. Although its inhibition promotes longevity across species, the effect of attenuated IGF1 signaling on cardiac aging remains controversial.
    METHODS: We performed a lifelong study to assess cardiac health and lifespan in 2 cardiomyocyte-specific transgenic mouse models with enhanced versus reduced IGF1 receptor (IGF1R) signaling. Male mice with human IGF1 receptor overexpression or dominant negative phosphoinositide 3-kinase mutation were examined at different life stages by echocardiography, invasive hemodynamics, and treadmill coupled to indirect calorimetry. In vitro assays included cardiac histology, mitochondrial respiration, ATP synthesis, autophagic flux, and targeted metabolome profiling, and immunoblots of key IGF1R downstream targets in mouse and human explanted failing and nonfailing hearts, as well.
    RESULTS: Young mice with increased IGF1R signaling exhibited superior cardiac function that progressively declined with aging in an accelerated fashion compared with wild-type animals, resulting in heart failure and a reduced lifespan. In contrast, mice with low cardiac IGF1R signaling exhibited inferior cardiac function early in life, but superior cardiac performance during aging, and increased maximum lifespan, as well. Mechanistically, the late-life detrimental effects of IGF1R activation correlated with suppressed autophagic flux and impaired oxidative phosphorylation in the heart. Low IGF1R activity consistently improved myocardial bioenergetics and function of the aging heart in an autophagy-dependent manner. In humans, failing hearts, but not those with compensated hypertrophy, displayed exaggerated IGF1R expression and signaling activity.
    CONCLUSIONS: Our findings indicate that the relationship between IGF1R signaling, and cardiac health is not linear, but rather biphasic. Hence, pharmacological inhibitors of the IGF1 pathway, albeit unsuitable for young individuals, might be worth considering in older adults.
    Keywords:  aging; autophagy; cardiomyopathies; insulin-like growth factor-1, mouse; mitochondria; phosphatidylinositol 3-kinases
    DOI:  https://doi.org/10.1161/CIRCULATIONAHA.122.059863
  12. Genes (Basel). 2022 Apr 27. pii: 778. [Epub ahead of print]13(5):
    Inferring Potential Cancer Driving Synonymous Variants.
    Zishuo Zeng, Yana Bromberg.
      Synonymous single nucleotide variants (sSNVs) are often considered functionally silent, but a few cases of cancer-causing sSNVs have been reported. From available databases, we collected four categories of sSNVs: germline, somatic in normal tissues, somatic in cancerous tissues, and putative cancer drivers. We found that screening sSNVs for recurrence among patients, conservation of the affected genomic position, and synVep prediction (synVep is a machine learning-based sSNV effect predictor) recovers cancer driver variants (termed proposed drivers) and previously unknown putative cancer genes. Of the 2.9 million somatic sSNVs found in the COSMIC database, we identified 2111 proposed cancer driver sSNVs. Of these, 326 sSNVs could be further tagged for possible RNA splicing effects, RNA structural changes, and affected RBP motifs. This list of proposed cancer driver sSNVs provides computational guidance in prioritizing the experimental evaluation of synonymous mutations found in cancers. Furthermore, our list of novel potential cancer genes, galvanized by synonymous mutations, may highlight yet unexplored cancer mechanisms.
    Keywords:  cancer drivers; sSNV; somatic variants; synonymous variants; variant functional impact
    DOI:  https://doi.org/10.3390/genes13050778
  13. Cancer Cell. 2022 May 25. pii: S1535-6108(22)00216-1. [Epub ahead of print]
    Redefining breast cancer subtypes to guide treatment prioritization and maximize response: Predictive biomarkers across 10 cancer therapies.
    I-SPY2 Investigators
      Using pre-treatment gene expression, protein/phosphoprotein, and clinical data from the I-SPY2 neoadjuvant platform trial (NCT01042379), we create alternative breast cancer subtypes incorporating tumor biology beyond clinical hormone receptor (HR) and human epidermal growth factor receptor-2 (HER2) status to better predict drug responses. We assess the predictive performance of mechanism-of-action biomarkers from ∼990 patients treated with 10 regimens targeting diverse biology. We explore >11 subtyping schemas and identify treatment-subtype pairs maximizing the pathologic complete response (pCR) rate over the population. The best performing schemas incorporate Immune, DNA repair, and HER2/Luminal phenotypes. Subsequent treatment allocation increases the overall pCR rate to 63% from 51% using HR/HER2-based treatment selection. pCR gains from reclassification and improved patient selection are highest in HR+ subsets (>15%). As new treatments are introduced, the subtyping schema determines the minimum response needed to show efficacy. This data platform provides an unprecedented resource and supports the usage of response-based subtypes to guide future treatment prioritization.
    Keywords:  DNA repair; Immune; Luminal; breast cancer; clinical trial; immunotherapy; multiple arms; platinum; response prediction; subtyping
    DOI:  https://doi.org/10.1016/j.ccell.2022.05.005
  14. Nat Commun. 2022 May 25. 13(1): 2904
    O-GlcNAc modification of leucyl-tRNA synthetase 1 integrates leucine and glucose availability to regulate mTORC1 and the metabolic fate of leucine.
    Kibum Kim, Hee Chan Yoo, Byung Gyu Kim, Sulhee Kim, Yulseung Sung, Ina Yoon, Ya Chun Yu, Seung Joon Park, Jong Hyun Kim, Kyungjae Myung, Kwang Yeon Hwang, Sunghoon Kim, Jung Min Han.
      All living organisms have the ability to sense nutrient levels to coordinate cellular metabolism. Despite the importance of nutrient-sensing pathways that detect the levels of amino acids and glucose, how the availability of these two types of nutrients is integrated is unclear. Here, we show that glucose availability regulates the central nutrient effector mTORC1 through intracellular leucine sensor leucyl-tRNA synthetase 1 (LARS1). Glucose starvation results in O-GlcNAcylation of LARS1 on residue S1042. This modification inhibits the interaction of LARS1 with RagD GTPase and reduces the affinity of LARS1 for leucine by promoting phosphorylation of its leucine-binding site by the autophagy-activating kinase ULK1, decreasing mTORC1 activity. The lack of LARS1 O-GlcNAcylation constitutively activates mTORC1, supporting its ability to sense leucine, and deregulates protein synthesis and leucine catabolism under glucose starvation. This work demonstrates that LARS1 integrates leucine and glucose availability to regulate mTORC1 and the metabolic fate of leucine.
    DOI:  https://doi.org/10.1038/s41467-022-30696-8
  15. Cell Signal. 2022 May 19. pii: S0898-6568(22)00118-8. [Epub ahead of print] 110356
    Differential impact of lipid raft depletion on platelet-derived growth factor (PDGF)-induced ERK1/2 MAP-kinase, SRC and AKT signaling.
    Erik Wåhlén, Frida Olsson, Ola Söderberg, Johan Lennartsson, Johan Heldin.
      It has become clear that lipid rafts functions as signaling hotspots connecting cell surface receptors to intracellular signaling pathways. However, the exact involvement of lipid rafts in receptor tyrosine kinase signaling is still poorly understood. In this study, we have analyzed platelet-derived growth factor (PDGF) receptor β (PDGFR-β) signaling in two different cell lines depleted of cholesterol and as a consequence, disruption of lipid rafts. Cholesterol depletion of BJ-hTERT fibroblasts using methyl-β-cyclodextrin (MβCD) did not affect PDGFR-β activation as measured by its tyrosine phosphorylation. However, we did observe a small reduction in AKT phosphorylation and a more robust decrease of ERK1/2 activation. In contrast, in the osteosarcoma cell line U2OS, we noticed a deficient receptor activation. Interestingly, in U2OS cells, the ERK1/2 pathway was unaffected, but instead AKT and SRC signaling was reduced. These results suggest that cell type specific wiring of signaling pathways can lead to differential sensitivity to cholesterol depletion. Furthermore, MβCD treatment had a much more pronounced morphological effect on U2OS compared to BJ-hTERT cells. This is consistent with a previous report claiming that cancer cells are more sensitive to cholesterol depletion than normal cells. Our data supports the possibility that cholesterol lowering drugs may impede tumor growth.
    Keywords:  BJ-hTERT and U2OS; Lipid rafts; Membrane rafts; MβCD; PDGFR
    DOI:  https://doi.org/10.1016/j.cellsig.2022.110356
  16. Curr Protoc. 2022 May;2(5): e435
    Assessing Cell Competition in Human Pluripotent Stem Cell (hPSC) Cultures.
    Christopher J Price, Ivana Barbaric.
      Cell-cell interactions are required for development and homeostasis in multicellular organisms from insects to mammals. A critical process governed by these interactions is cell competition, which functions throughout development to control tissue composition by eliminating cells that possess a lower fitness status than their neighbors. Human pluripotent stem cells (hPSCs) are a key biological tool in modeling human development and offer further potential as a source of clinically relevant cell populations for regenerative medicine applications. Recently, cell competition has been demonstrated in hPSC cultures and during induced pluripotent stem cell reprogramming. In turn, these findings suggest that hPSCs can be used as a tool to study and model cell-cell interactions during different stages of development and disease. Here, we provide a panel of protocols optimized for hPSCs to investigate the potential role that cell competition may have in determining the fate and composition of cell populations during culture. The protocols entail assessment of the competitive phenotype and the mode through which cell competition may lead to elimination of less-fit cells from mosaic cultures with fitter counterparts. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Electroporation of hPSCs to establish a fluorescent reference cell line Support Protocol 1: Single-cell dissociation of hPSCs Support Protocol 2: Single-cell cloning of fluorescently labeled hPSCs Basic Protocol 2: Separate culture and co-culture proliferation assays Basic Protocol 3: Assessing levels of apoptosis in hPSC cultures using flow cytometry Basic Protocol 4: Transwell assay Support Protocol 3: Immunohistochemistry and image quantification of cleaved caspase-3 Basic Protocol 5: Cell confrontation assay Basic Protocol 6: Cell compression assay Basic Protocol 7: Time-lapse imaging to assess mechanical extrusion.
    Keywords:  cell competition; culture-acquired genetic variants; fitness-sensing assays; fluorescent hPSC subline; human pluripotent stem cells (hPSCs); single-cell cloning hPSCs
    DOI:  https://doi.org/10.1002/cpz1.435
  17. iScience. 2022 Jun 17. 25(6): 104325
    c-JUN inhibits mTORC2 and glucose uptake to promote self-renewal and obesity.
    Raphael Serna, Ambika Ramrakhiani, Juan Carlos Hernandez, Chia-Lin Chen, Chad Nakagawa, Tatsuya Machida, Ratna B Ray, Xiaohang Zhan, Stanley M Tahara, Keigo Machida.
      Metabolic syndrome is associated with obesity, insulin resistance, and the risk of cancer. We tested whether oncogenic transcription factor c-JUN metabolically reprogrammed cells to induce obesity and cancer by reduction of glucose uptake, with promotion of the stemness phenotype leading to malignant transformation. Liquid alcohol, high-cholesterol, fat diet (HCFD), and isocaloric dextrin were fed to wild-type or experimental mice for 12 months to promote hepatocellular carcinoma (HCC). We demonstrated 40% of mice developed liver tumors after chronic HCFD feeding. Disruption of liver-specific c-Jun reduced tumor incidence 4-fold and improved insulin sensitivity. Overexpression of c-JUN downregulated RICTOR transcription, leading to inhibition of the mTORC2/AKT and glycolysis pathways. c-JUN inhibited GLUT1, 2, and 3 transactivation to suppress glucose uptake. Silencing of RICTOR or c-JUN overexpression promoted self-renewal ability. Taken together, c-JUN inhibited mTORC2 via RICTOR downregulation and inhibited glucose uptake via downregulation of glucose intake, leading to self-renewal and obesity.
    Keywords:  Biological sciences; Cancer; Cell biology; Diabetology; Endocrinology; Molecular biology
    DOI:  https://doi.org/10.1016/j.isci.2022.104325
  18. Biomolecules. 2022 Apr 20. pii: 612. [Epub ahead of print]12(5):
    Obesity, Diabetes, and Cancer: The Role of the Insulin/IGF Axis; Mechanisms and Clinical Implications.
    Andrea Morrione, Antonino Belfiore.
      This biomolecules Special Issue includes original research articles and reviews focusing on recent advances in the biology of the insulin-like growth factor (IGF) system [...].
    DOI:  https://doi.org/10.3390/biom12050612
  19. Trends Cell Biol. 2022 May 20. pii: S0962-8924(22)00116-7. [Epub ahead of print]
    The heterogeneity of cellular senescence: insights at the single-cell level.
    Rachel L Cohn, Nathan S Gasek, George A Kuchel, Ming Xu.
      Senescent cells are highly associated with aging and pathological conditions and could be targeted to slow the aging process. One commonly used marker to examine senescent cells in vivo is p16, which has led to important discoveries. Recent studies have also described new senescence markers beyond p16 and have highlighted the importance of investigating senescence heterogeneity in cell types and tissues. With the development of high-throughput technologies, such as single-cell RNA-seq and single-nucleus RNA-seq, we can examine senescent cells at the single-cell level and potentially uncover new markers. This review emphasizes that there is an urgent need to investigate senescence heterogeneity and discuss how this could be accomplished by using advanced technologies and sequencing datasets.
    Keywords:  RNA-sequencing; aging; p16; p21; senolytics
    DOI:  https://doi.org/10.1016/j.tcb.2022.04.011
  20. Dev Cell. 2022 May 15. pii: S1534-5807(22)00306-9. [Epub ahead of print]
    DRP1 levels determine the apoptotic threshold during embryonic differentiation through a mitophagy-dependent mechanism.
    Barbara Pernaute, Salvador Pérez-Montero, Juan Miguel Sánchez Nieto, Aida Di Gregorio, Ana Lima, Katerina Lawlor, Sarah Bowling, Gianmaria Liccardi, Alejandra Tomás, Pascal Meier, Hiromi Sesaki, Guy A Rutter, Ivana Barbaric, Tristan A Rodríguez.
      The changes that drive differentiation facilitate the emergence of abnormal cells that need to be removed before they contribute to further development or the germline. Consequently, in mice in the lead-up to gastrulation, ∼35% of embryonic cells are eliminated. This elimination is caused by hypersensitivity to apoptosis, but how it is regulated is poorly understood. Here, we show that upon exit of naive pluripotency, mouse embryonic stem cells lower their mitochondrial apoptotic threshold, and this increases their sensitivity to cell death. We demonstrate that this enhanced apoptotic response is induced by a decrease in mitochondrial fission due to a reduction in the activity of dynamin-related protein 1 (DRP1). Furthermore, we show that in naive pluripotent cells, DRP1 prevents apoptosis by promoting mitophagy. In contrast, during differentiation, reduced mitophagy levels facilitate apoptosis. Together, these results indicate that during early mammalian development, DRP1 regulation of mitophagy determines the apoptotic response.
    Keywords:  apoptosis; early development; embryonic stem cell differentiation; mitochondrial dynamics; mitophagy; pluripotency
    DOI:  https://doi.org/10.1016/j.devcel.2022.04.020
  21. Mol Cell. 2022 May 05. pii: S1097-2765(22)00327-6. [Epub ahead of print]
    Localization of a TORC1-eIF4F translation complex during CD8+ T cell activation drives divergent cell fate.
    Swantje Liedmann, Xueyan Liu, Clifford S Guy, Jeremy Chase Crawford, Diego A Rodriguez, Duygu Kuzuoğlu-Öztürk, Ao Guo, Katherine C Verbist, Jamshid Temirov, Mark J Chen, Davide Ruggero, Hui Zhang, Paul G Thomas, Douglas R Green.
      Activated CD8+ T lymphocytes differentiate into heterogeneous subsets. Using super-resolution imaging, we found that prior to the first division, dynein-dependent vesicular transport polarized active TORC1 toward the microtubule-organizing center (MTOC) at the proximal pole. This active TORC1 was physically associated with active eIF4F, required for the translation of c-myc mRNA. As a consequence, c-myc-translating polysomes polarized toward the cellular pole proximal to the immune synapse, resulting in localized c-myc translation. Upon division, the TORC1-eIF4A complex preferentially sorted to the proximal daughter cell, facilitating asymmetric c-Myc synthesis. Transient disruption of eIF4A activity at first division skewed long-term cell fate trajectories to memory-like function. Using a genetic barcoding approach, we found that first-division sister cells often displayed differences in transcriptional profiles that largely correlated with c-Myc and TORC1 target genes. Our findings provide mechanistic insights as to how distinct T cell fate trajectories can be established during the first division.
    Keywords:  CD8(+) T cells; STED; STORM; asymmetric cell division; c-Myc; cell fate; eIF4A; polarization; scRNA-seq; translation
    DOI:  https://doi.org/10.1016/j.molcel.2022.04.016
  22. Curr Top Dev Biol. 2022 ;pii: S0070-2153(21)00091-0. [Epub ahead of print]149 123-152
    Pulling back the curtain: The hidden functions of receptor tyrosine kinases in development.
    James F Clark, Philippe M Soriano.
      Receptor tyrosine kinases (RTKs) are a conserved superfamily of transmembrane growth factor receptors that drive numerous cellular processes during development and in the adult. Upon activation, multiple adaptors and signaling effector proteins are recruited to binding site motifs located within the intracellular domain of the RTK. These RTK-effector interactions drive subsequent intracellular signaling cascades involved in canonical RTK signaling. Genetic dissection has revealed that alleles of Fibroblast Growth Factor receptors (FGFRs) that lack all canonical RTK signaling still retain some kinase-dependent biological activity. Here we examine how genetic analysis can be used to understand the mechanism by which RTKs drive multiple developmental processes via canonical signaling while revealing noncanonical activities. Recent data from both FGFRs and other RTKs highlight potential noncanonical roles in cell adhesion and nuclear signaling. The data supporting such functions are discussed as are recent technologies that have the potential to provide valuable insight into the developmental significance of these noncanonical activities.
    Keywords:  Cell adhesion; Fibroblast growth factor signaling; Nuclear signaling; Receptor tyrosine kinases
    DOI:  https://doi.org/10.1016/bs.ctdb.2021.12.001
  23. J Proteome Res. 2022 May 24.
    In Vitro Kinase-to-Phosphosite Database (iKiP-DB) Predicts Kinase Activity in Phosphoproteomic Datasets.
    Tommaso Mari, Kirstin Mösbauer, Emanuel Wyler, Markus Landthaler, Christian Drosten, Matthias Selbach.
      Phosphoproteomics routinely quantifies changes in the levels of thousands of phosphorylation sites, but functional analysis of such data remains a major challenge. While databases like PhosphoSitePlus contain information about many phosphorylation sites, the vast majority of known sites is not assigned to any protein kinase. Assigning changes in the phosphoproteome to the activity of individual kinases therefore remains a key challenge. A recent large-scale study systematically identified in vitro substrates for most human protein kinases. Here, we reprocessed and filtered these data to generate an in vitro Kinase-to-Phosphosite database (iKiP-DB). We show that iKiP-DB can accurately predict changes in kinase activity in published phosphoproteomic data sets for both well-studied and poorly characterized kinases. We apply iKiP-DB to a newly generated phosphoproteomic analysis of SARS-CoV-2 infected human lung epithelial cells and provide evidence for coronavirus-induced changes in host cell kinase activity. In summary, we show that iKiP-DB is widely applicable to facilitate the functional analysis of phosphoproteomic data sets.
    Keywords:  SARS-CoV-2; kinase enrichment; mass spectrometry; phosphoproteomics; phosphosite annotations; phosphosites database; tandem mass tags
    DOI:  https://doi.org/10.1021/acs.jproteome.2c00198
  24. J Biol Chem. 2022 May 20. pii: S0021-9258(22)00498-7. [Epub ahead of print] 102058
    Phosphatidylinositol Phosphates Modulate Interactions between the StarD4 Sterol Trafficking Protein and Lipid Membranes.
    Xiaoxue Zhang, Hengyi Xie, David Iaea, George Khelashvili, Harel Weinstein, Frederick R Maxfield.
      There is substantial evidence for extensive non-vesicular sterol transport in cells. For example, lipid transfer by the steroidogenic acute regulator-related proteins (StarD) containing a StarT domain has been shown to involve several pathways of non-vesicular trafficking. Among the soluble StarT domain containing proteins, StarD4 is expressed in most tissues and has been shown to be an effective sterol transfer protein. However, it was unclear whether the lipid composition of donor or acceptor membranes played a role in modulating StarD4-mediated transport. Here we used fluorescence-based assays to demonstrate a phosphatidylinositol phosphate (PIP)-selective mechanism by which StarD4 can preferentially extract sterol from liposome membranes containing certain PIPs (especially, PI(4,5)P2 and to a lesser degree PI(3,5)P2). Monophosphorylated PIPs and other anionic lipids had a smaller effect on sterol transport. This enhancement of transport was less effective when the same PIPs were present in the acceptor membranes. Furthermore, using molecular dynamics simulations, we mapped the key interaction sites of StarD4 with PIP-containing membranes and identified residues that are important for this interaction and for accelerated sterol transport activity. We show that StarD4 recognizes membrane-specific PIPs through specific interaction with the geometry of the PIP head group as well as the surrounding membrane environment. Finally, we also observed that StarD4 can deform membranes upon longer incubations. Taken together, these results suggest a mechanism by which phosphatidylinositol phosphates modulate cholesterol transfer activity via StarD4.
    Keywords:  cholesterol‐binding protein; computer modeling; electron microscopy (EM); lipid transport; liposome; membrane lipid
    DOI:  https://doi.org/10.1016/j.jbc.2022.102058
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