bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2022‒04‒03
seventeen papers selected by
Ralitsa Radostinova Madsen
University College London Cancer Institute

  1. Breast Cancer Res. 2022 Apr 01. 24(1): 23
      BACKGROUND: PI3K signaling is frequently activated in breast cancer and is targeted by PI3K inhibitors. However, resistance of tumor cells to PI3K inhibition, often mediated by activated receptor tyrosine kinases, is commonly observed and reduces the potency of PI3K inhibitors. Therefore, new treatment strategies to overcome resistance to PI3K inhibitors are urgently needed to boost their efficacy. The phosphatase SHP2, which plays a crucial role in mediating signal transduction between receptor tyrosine kinases and both the PI3K and MAPK pathways, is a potential target for combination treatment.METHODS: We tested combinations of PI3K and SHP2 inhibitors in several experimental breast cancer models that are resistant to PI3K inhibition. Using cell culturing, biochemical and genetic approaches, we evaluated tumor cell proliferation and signaling output in cells treated with PI3K and SHP2 inhibitors.
    RESULTS: Combination treatment with PI3K and SHP2 inhibitors counteracted both acquired and intrinsic breast cancer cell resistance to PI3K inhibition that is mediated by activated receptor tyrosine kinases. Dual PI3K and SHP2 inhibition blocked proliferation and led to sustained inactivation of PI3K and MAPK signaling, where resistant cells rapidly re-activated these pathways upon PI3K inhibitor monotreatment. In addition, we demonstrate that overexpression of SHP2 induced resistance to PI3K inhibition, and that SHP2 was frequently activated during the development of PI3K inhibitor resistance after prolonged treatment of sensitive cells.
    CONCLUSIONS: Our results highlight the importance of SHP2 as a player in resistance to PI3K inhibitors. Combination treatment with PI3K and SHP2 inhibitors could pave the way for significant improvements in therapies for breast cancer.
    Keywords:  Breast cancer; Drug resistance; PI3K and MAPK signaling; SHP2; Targeted therapy
  2. Brain. 2022 Mar 31. pii: awab376. [Epub ahead of print]
      Focal malformations of cortical development including focal cortical dysplasia, hemimegalencephaly and megalencephaly, are a spectrum of neurodevelopmental disorders associated with brain overgrowth, cellular and architectural dysplasia, intractable epilepsy, autism and intellectual disability. Importantly, focal cortical dysplasia is the most common cause of focal intractable paediatric epilepsy. Gain and loss of function variants in the PI3K-AKT-MTOR pathway have been identified in this spectrum, with variable levels of mosaicism and tissue distribution. In this study, we performed deep molecular profiling of common PI3K-AKT-MTOR pathway variants in surgically resected tissues using droplet digital polymerase chain reaction (ddPCR), combined with analysis of key phenotype data. A total of 159 samples, including 124 brain tissue samples, were collected from 58 children with focal malformations of cortical development. We designed an ultra-sensitive and highly targeted molecular diagnostic panel using ddPCR for six mutational hotspots in three PI3K-AKT-MTOR pathway genes, namely PIK3CA (p.E542K, p.E545K, p.H1047R), AKT3 (p.E17K) and MTOR (p.S2215F, p.S2215Y). We quantified the level of mosaicism across all samples and correlated genotypes with key clinical, neuroimaging and histopathological data. Pathogenic variants were identified in 17 individuals, with an overall molecular solve rate of 29.31%. Variant allele fractions ranged from 0.14 to 22.67% across all mutation-positive samples. Our data show that pathogenic MTOR variants are mostly associated with focal cortical dysplasia, whereas pathogenic PIK3CA variants are more frequent in hemimegalencephaly. Further, the presence of one of these hotspot mutations correlated with earlier onset of epilepsy. However, levels of mosaicism did not correlate with the severity of the cortical malformation by neuroimaging or histopathology. Importantly, we could not identify these mutational hotspots in other types of surgically resected epileptic lesions (e.g. polymicrogyria or mesial temporal sclerosis) suggesting that PI3K-AKT-MTOR mutations are specifically causal in the focal cortical dysplasia-hemimegalencephaly spectrum. Finally, our data suggest that ultra-sensitive molecular profiling of the most common PI3K-AKT-MTOR mutations by targeted sequencing droplet digital polymerase chain reaction is an effective molecular approach for these disorders with a good diagnostic yield when paired with neuroimaging and histopathology.
    Keywords:  ddPCR; epilepsy; focal cortical dysplasia; hemimegalencephaly; mosaicism
  3. Nature. 2022 Mar 30.
      Oncogenic alterations to DNA are not transforming in all cellular contexts1,2. This may be due to pre-existing transcriptional programmes in the cell of origin. Here we define anatomic position as a major determinant of why cells respond to specific oncogenes. Cutaneous melanoma arises throughout the body, whereas the acral subtype arises on the palms of the hands, soles of the feet or under the nails3. We sequenced the DNA of cutaneous and acral melanomas from a large cohort of human patients and found a specific enrichment for BRAF mutations in cutaneous melanoma and enrichment for CRKL amplifications in acral melanoma. We modelled these changes in transgenic zebrafish models and found that CRKL-driven tumours formed predominantly in the fins of the fish. The fins are the evolutionary precursors to tetrapod limbs, indicating that melanocytes in these acral locations may be uniquely susceptible to CRKL. RNA profiling of these fin and limb melanocytes, when compared with body melanocytes, revealed a positional identity gene programme typified by posterior HOX13 genes. This positional gene programme synergized with CRKL to amplify insulin-like growth factor (IGF) signalling and drive tumours at acral sites. Abrogation of this CRKL-driven programme eliminated the anatomic specificity of acral melanoma. These data suggest that the anatomic position of the cell of origin endows it with a unique transcriptional state that makes it susceptible to only certain oncogenic insults.
  4. Cell Stem Cell. 2022 Mar 23. pii: S1934-5909(22)00099-6. [Epub ahead of print]
      Measuring cell identity in development, disease, and reprogramming is challenging as cell types and states are in continual transition. Here, we present Capybara, a computational tool to classify discrete cell identity and intermediate "hybrid" cell states, supporting a metric to quantify cell fate transition dynamics. We validate hybrid cells using experimental lineage tracing data to demonstrate the multi-lineage potential of these intermediate cell states. We apply Capybara to diagnose shortcomings in several cell engineering protocols, identifying hybrid states in cardiac reprogramming and off-target identities in motor neuron programming, which we alleviate by adding exogenous signaling factors. Further, we establish a putative in vivo correlate for induced endoderm progenitors. Together, these results showcase the utility of Capybara to dissect cell identity and fate transitions, prioritizing interventions to enhance the efficiency and fidelity of stem cell engineering.
    Keywords:  cell differentiation; cell reprogramming; cell-type classification; hybrid cells; single-cell analysis
  5. J Am Chem Soc. 2022 Mar 30.
      Covalent protein kinase inhibitors exploit currently noncatalytic cysteines in the adenosine 5'-triphosphate (ATP)-binding site via electrophiles directly appended to a reversible-inhibitor scaffold. Here, we delineate a path to target solvent-exposed cysteines at a distance >10 Å from an ATP-site-directed core module and produce potent covalent phosphoinositide 3-kinase α (PI3Kα) inhibitors. First, reactive warheads are used to reach out to Cys862 on PI3Kα, and second, enones are replaced with druglike warheads while linkers are optimized. The systematic investigation of intrinsic warhead reactivity (kchem), rate of covalent bond formation and proximity (kinact and reaction space volume Vr), and integration of structure data, kinetic and structural modeling, led to the guided identification of high-quality, covalent chemical probes. A novel stochastic approach provided direct access to the calculation of overall reaction rates as a function of kchem, kinact, Ki, and Vr, which was validated with compounds with varied linker lengths. X-ray crystallography, protein mass spectrometry (MS), and NanoBRET assays confirmed covalent bond formation of the acrylamide warhead and Cys862. In rat liver microsomes, compounds 19 and 22 outperformed the rapidly metabolized CNX-1351, the only known PI3Kα irreversible inhibitor. Washout experiments in cancer cell lines with mutated, constitutively activated PI3Kα showed a long-lasting inhibition of PI3Kα. In SKOV3 cells, compounds 19 and 22 revealed PI3Kβ-dependent signaling, which was sensitive to TGX221. Compounds 19 and 22 thus qualify as specific chemical probes to explore PI3Kα-selective signaling branches. The proposed approach is generally suited to develop covalent tools targeting distal, unexplored Cys residues in biologically active enzymes.
  6. Trends Neurosci. 2022 Mar 28. pii: S0166-2236(22)00052-2. [Epub ahead of print]
      Since its discovery over 100 years ago, insulin has been recognized as a key hormone in control of glucose homeostasis. Deficiencies of insulin signaling are central to diabetes and many other disorders. The brain is among the targets of insulin action, and insulin resistance is a major contributor to many diseases, including brain disorders. Here, we summarize key roles of insulin action in the brain and how this involves different brain cell types. Disordered brain insulin signaling can also contribute to neuropsychiatric diseases, affecting brain circuits involved in mood and cognition. Understanding of insulin signaling in different brain cell types/circuits and how these are altered in disease may lead to the development of new therapeutic approaches to these challenging disorders.
    Keywords:  Alzheimer’s diseases; astrocytes; depression; diabetes; insulin resistance; neurons
  7. Cell Death Discov. 2022 Mar 31. 8(1): 146
      Regulation of cell size is crucial for organ development. Insulin signaling regulates organ size by antagonizing the subgroup O of forkhead box transcription factor (Foxo) through 14-3-3 in Drosophila. However, mechanisms for controlling the level and the nuclear localization of Foxo in developing organs are not well understood. Here, we investigate the role of Drosophila Translationally controlled tumor protein (Tctp) and its interacting partner 14-3-3 in Foxo regulation during organ development. Foxo overexpression in the developing eye disc results in growth inhibition. We show that Tctp overexpression antagonizes the Foxo effect by downregulating the Foxo level in the eye disc. Foxo overexpression or knockdown of Tctp in the larval salivary gland results in reduced gland size, mainly due to reduced cell size by defects in endoreplication. Whereas 14-3-3ζ knockdown has a negligible effect, knockdown of 14-3-3ε mimics the effect of Foxo overexpression or Tctp knockdown, suggesting an isoform-specific role of 14-3-3. Unlike nuclear enrichment of the endogenous Foxo in the salivary gland, overexpressed Foxo protein is largely distributed in the cytoplasm, and this mislocalization is restored by Tctp overexpression. Opposite to the effect of Tctp overexpression, Tctp knockdown increases cytoplasmic Foxo levels while decreasing nuclear Foxo levels. Together, our data suggest that Tctp and 14-3-3ε play critical roles in cell growth by reducing cytoplasmic Foxo levels. Knockdown of human TCTP also elevates the level of cytoplasmic FOXO1 in HeLa cells, suggesting that human TCTP may have a conserved role in downregulating FOXO in human cells.
  8. Neuropeptides. 2022 Mar 18. pii: S0143-4179(22)00018-X. [Epub ahead of print]93 102243
      The analgesic efficacy of morphine can be affected by a variety of factors. Our previous studies demonstrated that chemokine (CXC motif) ligand 10 (CXCL10) could induce algesia directly and attenuate the analgesic effect produced by a single dose of morphine. However, the underlying mechanism remains unclear. In the present study, we aimed to further investigate the mechanism of CXCL10-mediated inhibition on morphine analgesic effect. According to our findings, recombinant CXCL10 protein (rmCXCL10) could increase the phosphorylation of serine-threonine kinase AKT reduced by morphine in spinal cord. Blocking AKT activation by phosphoinositide 3-kinase (PI3K) inhibitor could effectively attenuate CXCL10-induced algesia, and reverse the decrease of paw withdrawal thresholds caused by the co-administration of morphine and rmCXCL10. Furthermore, rmCXCL10 could enhance the spinal expression of pro-inflammatory cytokines, including TNF-α, IL-6, and IL-1β, which could be blocked by PI3K inhibitor. In summary, these findings suggest that PI3K-AKT signaling pathway mediates the effect of CXCL10 on the regulation of morphine analgesia and the release of cytokines in spinal cord. Our study provides a new insight into the mechanism of chemokine-relative pain regulation.
    Keywords:  AKT; CXCL10; Chemokine; Morphine; PI3K; Pain
  9. Methods Mol Biol. 2022 ;2488 183-206
      Fluorescent live cell time-lapse microscopy is steadily contributing to our better understanding of the relationship between cell signaling and fate. However, large volumes of time-series data generated in these experiments and the heterogenous nature of signaling responses due to cell-cell variability hinder the exploration of such datasets. The population averages insufficiently describe the dynamics, yet finding prototypic dynamic patterns that relate to different cell fates is difficult when mining thousands of time-series. Here we demonstrate a protocol where we identify such dynamic phenotypes in a population of PC-12 cells that respond to a range of sustained growth factor perturbations. We use Time-Course Inspector, a free R/Shiny web application to explore and cluster single-cell time-series.
    Keywords:  Cell-cell heterogeneity; Clustering; Computational biology; Data analysis; Signal processing; Signaling dynamics; Single-cell data; Time-series
  10. Nat Struct Mol Biol. 2022 Mar 31.
      Insulin receptor (IR) signaling controls multiple facets of animal physiology. Maximally four insulins bind to IR at two distinct sites, termed site-1 and site-2. However, the precise functional roles of each binding event during IR activation remain unresolved. Here, we showed that IR incompletely saturated with insulin predominantly forms an asymmetric conformation and exhibits partial activation. IR with one insulin bound adopts a Γ-shaped conformation. IR with two insulins bound assumes a Ƭ-shaped conformation. One insulin binds at site-1 and another simultaneously contacts both site-1 and site-2 in the Ƭ-shaped IR dimer. We further show that concurrent binding of four insulins to sites-1 and -2 prevents the formation of asymmetric IR and promotes the T-shaped symmetric, fully active state. Collectively, our results demonstrate how the synergistic binding of multiple insulins promotes optimal IR activation.
  11. Compr Psychoneuroendocrinol. 2020 Nov;pii: 100013. [Epub ahead of print]4
      Background/Objectives: Environmental enrichment (EE) is a physiological model to investigate brain-fat interactions. We previously discovered that EE activates the hypothalamic-sympathoneural adipocyte (HSA) axis via induction of brain-derived neurotrophic factor (BDNF), thus leading to sympathetic stimulation of white adipose tissue (WAT) and an anti-obesity phenotype. Here, we investigate whether PTEN acts as a downstream mediator of the HSA axis in the EE.Methods: Mice were housed in EE for 4- and 16-week periods to determine how EE regulates adipose PTEN. Hypothalamic injections of adeno-associated viral (AAV) vectors expressing BDNF and a dominant negative form of its receptor were performed to assess the role of the HSA axis in adipose PTEN upregulation. A β-blocker, propranolol, and a denervation agent, 6-hydroydopamine, were administered to assess sympathetic signaling in the observed EE-PTEN phenotype. To determine whether inducing PTEN is sufficient to reproduce certain EE adipose remodeling, we overexpressed PTEN in WAT using an AAV vector. To determine whether adipose PTEN is necessary for the EE-mediated reduction in adipocyte size, we injected a rAAV vector expressing Cre recombinase to the WAT of adult PTENflox mice and placed the mice in EE.
    Results: EE upregulated adipose PTEN expression, which was associated with suppression of AKT and ERK phosphorylation, increased hormone-sensitive lipase (HSL) phosphorylation, and reduced adiposity. PTEN regulation was found to be controlled by the HSA axis-with the hypothalamic BDNF acting as the upstream mediator-and dependent on sympathetic innervation. AAV-mediated adipose PTEN overexpression recapitulated EE-mediated adipose changes including suppression of AKT and ERK phosphorylation, increased HSL phosphorylation, and reduced adipose mass, whereas PTEN knockdown blocked the EE-induced reduction of adipocyte size.
    Conclusions: These data suggest that adipose PTEN responds to environmental stimuli and serves as downstream mediator of WAT remodeling in the EE paradigm, resulting in decreased adipose mass and decreased adipocyte size.
    Keywords:  AAV; PTEN; adipose tissue; environmental enrichment; lipolysis; sympathetic nervous system
  12. Cell Rep. 2022 Mar 29. pii: S2211-1247(22)00317-5. [Epub ahead of print]38(13): 110573
      Haploinsufficiency describes a phenomenon where one functioning allele is insufficient for a normal phenotype, underlying several human diseases. The effect of haploinsufficiency on human embryonic stem cells (hESC) has not been thoroughly studied. To establish a genome-wide loss-of-function screening for heterozygous mutations, we fuse normal haploid hESCs with a library of mutant haploid hESCs. We identify over 600 genes with a negative effect on hESC growth in a haploinsufficient manner and characterize them as genes showing less tolerance to mutations, conservation during evolution, and depletion from telomeres and X chromosome. Interestingly, a large fraction of these genes is associated with extracellular matrix and plasma membrane and enriched for genes within WNT and TGF-β pathways. We thus identify haploinsufficiency-related genes that show growth retardation in early embryonic cells, suggesting dosage-dependent phenotypes in hESCs. Overall, we construct a unique model for studying haploinsufficiency and identified important dosage-dependent pathways involved in hESC growth and survival.
    Keywords:  CP: Cell Biology; CP: Stem Cell Research; essential genes; genetic screening; haploinsufficiency; human embryonic stem cells; signaling pathways
  13. Trends Biochem Sci. 2022 Mar 28. pii: S0968-0004(22)00062-7. [Epub ahead of print]
      Protein kinase C (PKC) isozymes are maintained in a 'ready-to-go' but 'safe' autoinhibited conformation until second messenger binding unleashes an autoinhibitory pseudosubstrate to allow substrate phosphorylation. However, to gain this 'ready-to-go' conformation, PKC must be processed by a series of complex priming phosphorylations, the mechanism of which was enigmatic until now. Recent findings snapped the pieces of the phosphorylation puzzle into place to unveil a process that involves a newly described motif (TOR interaction motif, TIM), a well-described kinase [mechanistic target of rapamycin complex 2 (mTORC2)], and an often-used mechanism (autophosphorylation) to prime PKC to signal. This review highlights new insights into how phosphorylation controls PKC and discusses them in the context of common mechanisms for AGC kinase regulation by phosphorylation and autophosphorylation.
    Keywords:  PKC; autophosphorylation; mTORC2; phosphorylation
  14. Nat Rev Mol Cell Biol. 2022 Apr 01.
      Mechanical signalling affects multiple biological processes during development and in adult organisms, including cell fate transitions, cell migration, morphogenesis and immune responses. Here, we review recent insights into the mechanisms and functions of two main routes of mechanical signalling: outside-in mechanical signalling, such as mechanosensing of substrate properties or shear stresses; and mechanical signalling regulated by the physical properties of the cell surface itself. We discuss examples of how these two classes of mechanical signalling regulate stem cell function, as well as developmental processes in vivo. We also discuss how cell surface mechanics affects intracellular signalling and, in turn, how intracellular signalling controls cell surface mechanics, generating feedback into the regulation of mechanosensing. The cooperation between mechanosensing, intracellular signalling and cell surface mechanics has a profound impact on biological processes. We discuss here our understanding of how these three elements interact to regulate stem cell fate and development.
  15. Sci Transl Med. 2022 Mar 30. 14(638): eabl6328
      Cyclic adenosine 3',5'-monophosphate (cAMP)-elevating agents, such as β2-adrenergic receptor (β2-AR) agonists and phosphodiesterase (PDE) inhibitors, remain a mainstay in the treatment of obstructive respiratory diseases, conditions characterized by airway constriction, inflammation, and mucus hypersecretion. However, their clinical use is limited by unwanted side effects because of unrestricted cAMP elevation in the airways and in distant organs. Here, we identified the A-kinase anchoring protein phosphoinositide 3-kinase γ (PI3Kγ) as a critical regulator of a discrete cAMP signaling microdomain activated by β2-ARs in airway structural and inflammatory cells. Displacement of the PI3Kγ-anchored pool of protein kinase A (PKA) by an inhaled, cell-permeable, PI3Kγ mimetic peptide (PI3Kγ MP) inhibited a pool of subcortical PDE4B and PDE4D and safely increased cAMP in the lungs, leading to airway smooth muscle relaxation and reduced neutrophil infiltration in a murine model of asthma. In human bronchial epithelial cells, PI3Kγ MP induced unexpected cAMP and PKA elevations restricted to the vicinity of the cystic fibrosis transmembrane conductance regulator (CFTR), the ion channel controlling mucus hydration that is mutated in cystic fibrosis (CF). PI3Kγ MP promoted the phosphorylation of wild-type CFTR on serine-737, triggering channel gating, and rescued the function of F508del-CFTR, the most prevalent CF mutant, by enhancing the effects of existing CFTR modulators. These results unveil PI3Kγ as the regulator of a β2-AR/cAMP microdomain central to smooth muscle contraction, immune cell activation, and epithelial fluid secretion in the airways, suggesting the use of a PI3Kγ MP for compartment-restricted, therapeutic cAMP elevation in chronic obstructive respiratory diseases.
  16. Nat Commun. 2022 Mar 29. 13(1): 1666
      Species-specific differences control cancer risk across orders of magnitude variation in body size and lifespan, e.g., by varying the copy numbers of tumor suppressor genes. It is unclear, however, how different tissues within an organism can control somatic evolution despite being subject to markedly different constraints, but sharing the same genome. Hierarchical differentiation, characteristic of self-renewing tissues, can restrain somatic evolution both by limiting divisional load, thereby reducing mutation accumulation, and by increasing cells' commitment to differentiation, which can "wash out" mutants. Here, we explore the organization of hierarchical tissues that have evolved to limit their lifetime incidence of cancer. Estimating the likelihood of cancer in the presence of mutations that enhance self-proliferation, we demonstrate that a trade-off exists between mutation accumulation and the strength of washing out. Our results explain differences in the organization of widely different hierarchical tissues, such as colon and blood.
  17. Nat Commun. 2022 Apr 01. 13(1): 1768
      Myeloid cells play key roles in cancer immune suppression and tumor progression. In response to tumor derived factors, circulating monocytes and granulocytes extravasate into the tumor parenchyma where they stimulate angiogenesis, immune suppression and tumor progression. Chemokines, cytokines and interleukins stimulate PI3Kγ-mediated Rap1 activation, leading to conformational changes in integrin α4β1 that promote myeloid cell extravasation and tumor inflammation Here we show that PI3Kγ activates a high molecular weight form of myosin light chain kinase, MLCK210, that promotes myosin-dependent Rap1 GTP loading, leading to integrin α4β1 activation. Genetic or pharmacological inhibition of MLCK210 suppresses integrin α4β1 activation, as well as tumor inflammation and progression. These results demonstrate a critical role for myeloid cell MLCK210 in tumor inflammation and serve as basis for the development of alternative approaches to develop immune oncology therapeutics.