bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2023‒08‒13
thirteen papers selected by
Ralitsa Radostinova Madsen

  1. Mol Cell. 2023 Aug 08. pii: S1097-2765(23)00558-0. [Epub ahead of print]
      The PIP3/PI3K network is a central regulator of metabolism and is frequently activated in cancer, commonly by loss of the PIP3/PI(3,4)P2 phosphatase, PTEN. Despite huge research investment, the drivers of the PI3K network in normal tissues and how they adapt to overactivation are unclear. We find that in healthy mouse prostate PI3K activity is driven by RTK/IRS signaling and constrained by pathway feedback. In the absence of PTEN, the network is dramatically remodeled. A poorly understood YXXM- and PIP3/PI(3,4)P2-binding PH domain-containing adaptor, PLEKHS1, became the dominant activator and was required to sustain PIP3, AKT phosphorylation, and growth in PTEN-null prostate. This was because PLEKHS1 evaded pathway-feedback and experienced enhanced PI3K- and Src-family kinase-dependent phosphorylation of Y258XXM, eliciting PI3K activation. hPLEKHS1 mRNA and activating Y419 phosphorylation of hSrc correlated with PI3K pathway activity in human prostate cancers. We propose that in PTEN-null cells receptor-independent, Src-dependent tyrosine phosphorylation of PLEKHS1 creates positive feedback that escapes homeostasis, drives PIP3 signaling, and supports tumor progression.
    Keywords:  IRS1; PI3K; PLEKHS1; PTEN; Src-family kinase; prostate
  2. Nat Cell Biol. 2023 Aug 10.
      Cell growth is regulated by the mammalian/mechanistic target of rapamycin complex 1 (mTORC1), which functions both as a nutrient sensor and a master controller of virtually all biosynthetic pathways. This ensures that cells are metabolically active only when conditions are optimal for growth. Notably, although mTORC1 is known to regulate fatty acid biosynthesis, how and whether the cellular lipid biosynthetic capacity signals back to fine-tune mTORC1 activity remains poorly understood. Here we show that mTORC1 senses the capacity of a cell to synthesise fatty acids by detecting the levels of malonyl-CoA, an intermediate of this biosynthetic pathway. We find that, in both yeast and mammalian cells, this regulation is direct, with malonyl-CoA binding to the mTOR catalytic pocket and acting as a specific ATP-competitive inhibitor. When fatty acid synthase (FASN) is downregulated/inhibited, elevated malonyl-CoA levels are channelled to proximal mTOR molecules that form direct protein-protein interactions with acetyl-CoA carboxylase 1 (ACC1) and FASN. Our findings represent a conserved and unique homeostatic mechanism whereby impaired fatty acid biogenesis leads to reduced mTORC1 activity to coordinately link this metabolic pathway to the overall cellular biosynthetic output. Moreover, they reveal the existence of a physiological metabolite that directly inhibits the activity of a signalling kinase in mammalian cells by competing with ATP for binding.
  3. Elife. 2023 Aug 07. pii: e85444. [Epub ahead of print]12
      Multiple signaling pathways regulate the kinase GSK3β by inhibitory phosphorylation at Ser9, which then occupies the GSK3β priming pocket and blocks substrate binding. Since this mechanism should affect GSK3β activity towards all primed substrates, it is unclear why Ser9 phosphorylation does not affect other GSK3β-dependent pathways, such as Wnt signaling. We used biochemical reconstitution and cell culture assays to evaluate how Wnt-associated GSK3β is insulated from cross-activation by other signals. We found that the Wnt-specific scaffold protein Axin allosterically protects GSK3β from phosphorylation at Ser9 by upstream kinases, which prevents accumulation of pS9-GSK3β in the Axin-GSK3β complex. Scaffold proteins that protect bound proteins from alternative pathway reactions could provide a general mechanism to insulate signaling pathways from improper crosstalk.
    Keywords:  biochemistry; cell biology; chemical biology; human
  4. Proc Natl Acad Sci U S A. 2023 08 15. 120(33): e2303010120
      The regulation of gene expression through histone posttranslational modifications plays a crucial role in breast cancer progression. However, the molecular mechanisms underlying the contribution of histone modification to tumor initiation remain unclear. To gain a deeper understanding of the role of the histone modifier Enhancer of Zeste homology 2 (Ezh2) in the early stages of mammary tumor progression, we employed an inducible mammary organoid system bearing conditional Ezh2 alleles that faithfully recapitulates key events of luminal B breast cancer initiation. We showed that the loss of Ezh2 severely impairs oncogene-induced organoid growth, with Ezh2-deficient organoids maintaining a polarized epithelial phenotype. Transcriptomic profiling showed that Ezh2-deficient mammary epithelial cells up-regulated the expression of negative regulators of Wnt signaling and down-regulated genes involved in mTORC1 (mechanistic target of rapamycin complex 1) signaling. We identified Sfrp1, a Wnt signaling suppressor, as an Ezh2 target gene that is derepressed and expressed in Ezh2-deficient epithelium. Furthermore, an analysis of breast cancer data revealed that Sfrp1 expression was associated with favorable clinical outcomes in luminal B breast cancer patients. Finally, we confirmed that targeting Ezh2 impairs mTORC1 activity through an indirect mechanism that up-regulates the expression of the tumor suppressor Pten. These findings indicate that Ezh2 integrates the mTORC1 and Wnt signaling pathways during early mammary tumor progression, arguing that inhibiting Ezh2 or therapeutically targeting Ezh2-dependent programs could be beneficial for the treatment of early-stage luminal B breast cancer.
    Keywords:  EZH2; Wnt signaling; breast cancer; mTOR signaling; tumor initiation
  5. bioRxiv. 2023 Jul 24. pii: 2023.07.23.550235. [Epub ahead of print]
      Glycolysis is a fundamental cellular process, yet its regulatory mechanisms remain incompletely understood. Here, we show that a subset of glucose transporter 1 (GLUT1/SLC2A1) co-endocytoses with platelet-derived growth factor (PDGF) receptor (PDGFR) upon PDGF-stimulation. Furthermore, multiple glycolytic enzymes localize to these endocytosed PDGFR/GLUT1-containing vesicles adjacent to mitochondria. Contrary to current models, which emphasize the importance of glucose transporters on the cell surface, we find that PDGF-stimulated glucose uptake depends on receptor/transporter endocytosis. Our results suggest that growth factors generate glucose-loaded endocytic vesicles that deliver glucose to the glycolytic machinery in proximity to mitochondria, and argue for a new layer of regulation for glycolytic control governed by cellular membrane dynamics.
  6. EMBO Rep. 2023 Aug 07. e56380
      Oxidative phosphorylation and glycolysis are the dominant ATP-generating pathways in mammalian metabolism. The balance between these two pathways is often shifted to execute cell-specific functions in response to stimuli that promote activation, proliferation, or differentiation. However, measurement of these metabolic switches has remained mostly qualitative, making it difficult to discriminate between healthy, physiological changes in energy transduction or compensatory responses due to metabolic dysfunction. We therefore present a broadly applicable method to calculate ATP production rates from oxidative phosphorylation and glycolysis using Seahorse XF Analyzer data and empirical conversion factors. We quantify the bioenergetic changes observed during macrophage polarization as well as cancer cell adaptation to in vitro culture conditions. Additionally, we detect substantive changes in ATP utilization upon neuronal depolarization and T cell receptor activation that are not evident from steady-state ATP measurements. This method generates a single readout that allows the direct comparison of ATP produced from oxidative phosphorylation and glycolysis in live cells. Additionally, the manuscript provides a framework for tailoring the calculations to specific cell systems or experimental conditions.
    Keywords:  ATP; ECAR; Seahorse XF Analyzer; glycolysis; oxidative phosphorylation
  7. Nat Commun. 2023 08 09. 14(1): 4816
      Cholesterol biosynthesis is a highly regulated, oxygen-dependent pathway, vital for cell membrane integrity and growth. In fungi, the dependency on oxygen for sterol production has resulted in a shared transcriptional response, resembling prolyl hydroxylation of Hypoxia Inducible Factors (HIFs) in metazoans. Whether an analogous metazoan pathway exists is unknown. Here, we identify Sterol Regulatory Element Binding Protein 2 (SREBP2), the key transcription factor driving sterol production in mammals, as an oxygen-sensitive regulator of cholesterol synthesis. SREBP2 degradation in hypoxia overrides the normal sterol-sensing response, and is HIF independent. We identify MARCHF6, through its NADPH-mediated activation in hypoxia, as the main ubiquitin ligase controlling SREBP2 stability. Hypoxia-mediated degradation of SREBP2 protects cells from statin-induced cell death by forcing cells to rely on exogenous cholesterol uptake, explaining why many solid organ tumours become auxotrophic for cholesterol. Our findings therefore uncover an oxygen-sensitive pathway for governing cholesterol synthesis through regulated SREBP2-dependent protein degradation.
  8. Sci Signal. 2023 08 08. 16(797): eadk1008
      Fasting activates mTORC2 to stimulate mitochondrial fission and support mitochondrial respiration.
  9. Nat Biotechnol. 2023 Aug 10.
      Although single-nucleotide variants (SNVs) make up the majority of cancer-associated genetic changes and have been comprehensively catalogued, little is known about their impact on tumor initiation and progression. To enable the functional interrogation of cancer-associated SNVs, we developed a mouse system for temporal and regulatable in vivo base editing. The inducible base editing (iBE) mouse carries a single expression-optimized cytosine base editor transgene under the control of a tetracycline response element and enables robust, doxycycline-dependent expression across a broad range of tissues in vivo. Combined with plasmid-based or synthetic guide RNAs, iBE drives efficient engineering of individual or multiple SNVs in intestinal, lung and pancreatic organoids. Temporal regulation of base editor activity allows controlled sequential genome editing ex vivo and in vivo, and delivery of sgRNAs directly to target tissues facilitates generation of in situ preclinical cancer models.
  10. Proc Natl Acad Sci U S A. 2023 08 15. 120(33): e2203828120
      Cellular omics such as single-cell genomics, proteomics, and microbiomics allow the characterization of tissue and microbial community composition, which can be compared between conditions to identify biological drivers. This strategy has been critical to revealing markers of disease progression, such as cancer and pathogen infection. A dedicated statistical method for differential variability analysis is lacking for cellular omics data, and existing methods for differential composition analysis do not model some compositional data properties, suggesting there is room to improve model performance. Here, we introduce sccomp, a method for differential composition and variability analyses that jointly models data count distribution, compositionality, group-specific variability, and proportion mean-variability association, being aware of outliers. sccomp provides a comprehensive analysis framework that offers realistic data simulation and cross-study knowledge transfer. Here, we demonstrate that mean-variability association is ubiquitous across technologies, highlighting the inadequacy of the very popular Dirichlet-multinomial distribution. We show that sccomp accurately fits experimental data, significantly improving performance over state-of-the-art algorithms. Using sccomp, we identified differential constraints and composition in the microenvironment of primary breast cancer.
    Keywords:  cell-type proportion; compositional; microbiome; single-cell; variability
  11. Front Cell Dev Biol. 2023 ;11 1236553
      Protocols to differentiate human pluripotent stem cells have advanced in terms of cell type specificity and tissue-level complexity over the past 2 decades, which has facilitated human disease modeling in the most relevant cell types. The ability to generate induced PSCs (iPSCs) from patients further enables the study of disease mutations in an appropriate cellular context to reveal the mechanisms that underlie disease etiology and progression. As iPSC-derived disease models have improved in robustness and scale, they have also been adopted more widely for use in drug screens to discover new therapies and therapeutic targets. Advancement in genome editing technologies, in particular the discovery of CRISPR-Cas9, has further allowed for rapid development of iPSCs containing disease-causing mutations. CRISPR-Cas9 technologies have now evolved beyond creating single gene edits, aided by the fusion of inhibitory (CRISPRi) or activation (CRISPRa) domains to a catalytically dead Cas9 protein, enabling inhibition or activation of endogenous gene loci. These tools have been used in CRISPR knockout, CRISPRi, or CRISPRa screens to identify genetic modifiers that synergize or antagonize with disease mutations in a systematic and unbiased manner, resulting in identification of disease mechanisms and discovery of new therapeutic targets to accelerate drug discovery research. However, many technical challenges remain when applying large-scale functional genomics approaches to differentiated PSC populations. Here we review current technologies in the field of iPSC disease modeling and CRISPR-based functional genomics screens and practical considerations for implementation across a range of modalities, applications, and disease areas, as well as explore CRISPR screens that have been performed in iPSC models to-date and the insights and therapies these screens have produced.
    Keywords:  CRISPR screening; drug discovery; functional genomics; human disease; iPSC (induced pluripotent stem cell); iPSC-derived models
  12. Annu Rev Genet. 2023 Aug 10.
      Assigning functions to genes and learning how to control their expression are part of the foundation of cell biology and therapeutic development. An efficient and unbiased method to accomplish this is genetic screening, which historically required laborious clone generation and phenotyping and is still limited by scale today. The rapid technological progress on modulating gene function with CRISPR-Cas and measuring it in individual cells has now relaxed the major experimental constraints and enabled pooled screening with complex readouts from single cells. Here, we review the principles and practical considerations for pooled single-cell CRISPR screening. We discuss perturbation strategies, experimental model systems, matching the perturbation to the individual cells, reading out cell phenotypes, and data analysis. Our focus is on single-cell RNA sequencing and cell sorting-based readouts, including image-enabled cell sorting. We expect this transformative approach to fuel biomedical research for the next several decades. Expected final online publication date for the Annual Review of Genetics, Volume 57 is November 2023. Please see for revised estimates.
  13. Methods Mol Biol. 2023 ;2706 97-124
      Kinases represent one of the most therapeutically tractable targets for drug discovery in the twenty-first century. However, confirming engagement and achieving intracellular kinase selectivity for small-molecule kinase inhibitors can represent noteworthy challenges. The NanoBRETTM platform enables broad-spectrum live-cell kinase selectivity profiling in most laboratory settings, without advanced instrumentation or expertise. However, the prototype workflow for this selectivity profiling is currently limited to manual liquid handling and 96-well plates. Herein, we describe a scalable workflow with automation and acoustic dispensing, thus dramatically improving the throughput. Such adaptations enable profiling of larger compound sets against 192 full-length protein kinases in live cells, with statistical robustness supporting quantitative analysis.
    Keywords:  BRET; High-Throughput Screening (HTS); Kinase profiling; Live-cell target engagement; NanoBRETTM