bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2022‒07‒24
eighteen papers selected by
Ralitsa Radostinova Madsen
University College London Cancer Institute

  1. Adv Ther. 2022 Jul 20.
      PIK3CA-related overgrowth spectrum (PROS) disorders are caused by somatic, gain-of-function mutations in PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha) that result in hyperactivation of the phosphatidylinositol-3-kinase (PI3K) signaling pathway. PROS encompasses a broad spectrum of overlapping phenotypes that vary considerably in their severity and tissue distribution, leading to different and complex experiences for affected children and their families. The parent of a child with the PROS disorder megalencephaly-capillary malformation (MCAP) coauthored this article. MCAP is characterized by significant neurological involvement, and she describes personal experiences with this condition, including delays associated with obtaining a correct diagnosis, finding an experienced care team, challenges with schooling, medical complications, and the ongoing emotional and financial impacts on their lives. A physician perspective, which reinforces the challenges faced by the young child and his family, is provided by a clinician and researcher specializing in PROS disorders with central nervous system involvement. The physician reviews the mechanism of disease, some of the challenges in accurately diagnosing PROS conditions, disease-related complications, current treatment options and their limitations, and emerging therapeutic options including ongoing clinical trials. Our objective is to share these experiences and insights to benefit patients with PROS disorders, their families, and health care professionals involved with caring for patients with PROS.
    Keywords:  Megalencephaly-capillary malformation; Mosaicism; PI3K; PIK3CA; PROS
  2. Front Cell Dev Biol. 2022 ;10 928210
      Pregnancy requires adaptations in maternal metabolism to support fetal growth. The phosphoinositol-3-kinase (PI3K) signalling pathway controls multiple biological processes and defects in this pathway are linked to metabolic disorders including insulin resistance and glucose intolerance in non-pregnant animals. However, relatively little is known about the contribution of PI3K signalling to the maternal metabolic adaptations during pregnancy. Using mice with partial inactivation of the PI3K isoform, p110α (due to a heterozygous dominant negative mutation; Pik3ca-D933A), the effects of impaired PI3K-p110α signalling on glucose and insulin handling were examined in the pregnant and non-pregnant states and related to the morphological, molecular, and mitochondrial changes in key metabolic organs. The results show that non-pregnant mice lacking PI3K-p110α are glucose intolerant but exhibit compensatory increases in pancreatic glucose-stimulated insulin release and adipose tissue mitochondrial respiratory capacity and fatty acid oxidation. However, in pregnancy, mutant mice failed to show the normal increment in glucose intolerance and pancreatic β-cell mass observed in wild-type pregnant dams and exhibited further enhanced adipose tissue mitochondrial respiratory capacity. These maladaptations in pregnant mutant mice were associated with fetal growth restriction. Hence, PI3K-p110α is a key regulator of metabolic adaptations that support fetal growth during normal pregnancy.
    Keywords:  PI3K; metabolism; mitochondria; pregnancy; signalling
  3. Comput Biol Chem. 2022 Jul 08. pii: S1476-9271(22)00106-2. [Epub ahead of print]99 107726
      PI3K pathway is heavily emphasized in cancer where PIK3CA, which encodes for the p110α subunit of PI3Kα, presents itself as the second most common mutated gene. A lot of effort has been put in developing PI3K inhibitors, opening promising avenues for the treatment of cancer. Among these, PI3Kα specific inhibitor alpelisib was approved by FDA for breast cancer and other α-isoform specific inhibitors such as inavolisib and serabelisib reached clinical trials. However, the mode of action of these inhibitors on mutated PI3Kα and how they interact with mutant structures has not been fully elucidated yet. In this study, we are revealing the calculated interactions and binding affinities of these inhibitors within the context of PIK3CA hotspot mutations (E542K, E545K and H1047R) by employing molecular dynamics (MD) simulations. We performed principal component analysis to understand the motions of the protein complex during our simulations and also checked the correlated motions of all amino acids. Binding affinity calculations with MM-PBSA confirmed the consistent binding of alpelisib across mutations and revealed relatively higher affinities for inavolisib towards wild-type and H1047R mutant structures in comparison to other inhibitors. On the other hand, E542K mutation significantly impaired the interaction of inavolisib and serabelisib with PI3Kα. We also investigated the structural relationship of the natural ligand ATP with PI3Kα, and interestingly realized a significant reduction in binding affinity for the mutants, with potentially unexpected implications on the mechanisms that render these mutations oncogenic. Moreover, correlated motions of all residues were generally higher for ATP except the H1047R mutation which exhibited a distinguishable reduction. The results presented here could be guiding for pre-clinical and clinical studies of personalized medicine where individual mutations are a strong consideration point.
    Keywords:  ATP; Alpelisib; E542K; E545K; H1047R; Inavolisib; Molecular dynamics; PI3K; PIK3CA; Serabelisib
  4. Sci Signal. 2022 Jul 19. 15(743): eabl9169
      The integrin lymphocyte function-associated antigen 1 (LFA-1) helps to coordinate the migration, adhesion, and activation of T cells through interactions with intercellular adhesion molecule 1 (ICAM-1) and ICAM-2. LFA-1 is activated during the engagement of chemokine receptors and the T cell receptor (TCR) through inside-out signaling, a process that is partially mediated by phosphoinositide 3-kinase (PI3K) and its product phosphatidylinositol 3,4,5-trisphosphate (PIP3). To evaluate potential roles of PI3K in LFA-1 activation, we designed a library of CRISPR/single guide RNAs targeting known and potential PIP3-binding proteins and screened for effects on the ability of primary mouse T cells to bind to ICAM-1. We identified multiple proteins that regulated the binding of LFA-1 to ICAM-1, including the Rap1 and Ras GTPase-activating protein RASA3. We found that RASA3 suppressed LFA-1 activation in T cells, that its expression was rapidly reduced upon T cell activation, and that its activity was inhibited by PI3K. Loss of RASA3 in T cells led to increased Rap1 activation, defective lymph node entry and egress, and impaired responses to T-dependent immunization in mice. Our results reveal a critical role for RASA3 in T cell migration, homeostasis, and function.
  5. Nature. 2022 Jul 20.
      Mechanistic target of rapamycin complex 1 (mTORC1) regulates cell growth and metabolism in response to multiple nutrients, including the essential amino acid leucine1. Recent work in cultured mammalian cells established the Sestrins as leucine-binding proteins that inhibit mTORC1 signalling during leucine deprivation2,3, but their role in the organismal response to dietary leucine remains elusive. Here we find that Sestrin-null flies (Sesn-/-) fail to inhibit mTORC1 or activate autophagy after acute leucine starvation and have impaired development and a shortened lifespan on a low-leucine diet. Knock-in flies expressing a leucine-binding-deficient Sestrin mutant (SesnL431E) have reduced, leucine-insensitive mTORC1 activity. Notably, we find that flies can discriminate between food with or without leucine, and preferentially feed and lay progeny on leucine-containing food. This preference depends on Sestrin and its capacity to bind leucine. Leucine regulates mTORC1 activity in glial cells, and knockdown of Sesn in these cells reduces the ability of flies to detect leucine-free food. Thus, nutrient sensing by mTORC1 is necessary for flies not only to adapt to, but also to detect, a diet deficient in an essential nutrient.
  6. Cancer Res. 2022 Jul 22. pii: CAN-22-0121. [Epub ahead of print]
      The mechanistic target of rapamycin (mTOR) is a key regulator of cell growth that integrates growth factor signaling and nutrient availability and is a downstream effector of oncogenic receptor tyrosine kinases (RTKs) and PI3K/Akt signaling. Thus, activating mTOR mutations would be expected to enhance growth in many tumor types. However, tumor sequencing data has shown that mTOR mutations are enriched only in renal clear cell carcinoma, a clinically hypervascular tumor unlikely to be constrained by nutrient availability. To further define this cancer type-specific restriction, we studied activating mutations in mTOR. All mTOR mutants tested enhanced growth in a cell type agnostic manner under nutrient-replete conditions but were detrimental to cell survival in nutrient-poor conditions. Consistently, analysis of tumor data demonstrated that oncogenic mutations in the nutrient-sensing arm of the mTOR pathway display a similar phenotype and were exceedingly rare in human cancers of all types. Together, these data suggest that maintaining the ability to turn off mTOR signaling in response to changing nutrient availability is retained in most naturally occurring tumors.
  7. Cell Rep. 2022 Jul 19. pii: S2211-1247(22)00931-7. [Epub ahead of print]40(3): 111125
      PTEN and LKB1 are intimately associated with gastrointestinal tumorigenesis. Mutations of PTEN or LKB1 lead to Cowden syndrome and Peutz-Jeghers syndrome characterized by development of gastrointestinal polyps. However, the cells of origin of these polyps and underlying mechanism remain unclear. Here, we reveal that PTEN or LKB1 deficiency in Gli1+ gut mesenchymal cells, but not intestinal epithelium, drives polyp formation histologically resembling polyposis in human patients. Mechanistically, although PTEN and LKB1 converge to regulate mTOR/AKT signaling in various tumor contexts, we find that mTOR is essential for PTEN-deletion-induced polyp formation but is largely dispensable for polyposis induced by mesenchymal LKB1 deficiency. Altogether, our studies identify Gli1-expressing mesenchymal cells as a common cell of origin for polyposis associated with PTEN and LKB1 and reveal their engagement of different downstream pathways in gut mesenchyme to suppress gastrointestinal tumorigenesis.
    Keywords:  AKT; CP: Cell biology; Gli1; LKB1; PTEN; gastrointestinal polyposis; intestinal mesenchyme; mTOR; tumorigenesis
  8. Sci Adv. 2022 Jul 08. 8(27): eabo0322
      Progerin, a product of LMNA mutation, leads to multiple nuclear abnormalities in patients with Hutchinson-Gilford progeria syndrome (HGPS), a devastating premature aging disorder. Progerin also accumulates during physiological aging. Here, we demonstrate that impaired insulin-like growth factor 1 receptor (IGF-1R)/Akt signaling pathway results in severe growth retardation and premature aging in Zmpste24-/- mice, a mouse model of progeria. Mechanistically, progerin mislocalizes outside of the nucleus, interacts with the IGF-1R, and down-regulates its expression, leading to inhibited mitochondrial respiration, retarded cell growth, and accelerated cellular senescence. Pharmacological treatment with the PTEN (phosphatase and tensin homolog deleted on chromosome 10) inhibitor bpV (HOpic) increases Akt activity and improves multiple abnormalities in Zmpste24-deficient mice. These findings provide previously unidentified insights into the role of progerin in regulating the IGF-1R/Akt signaling in HGPS and might be useful for treating LMNA-associated progeroid disorders.
  9. J Biol Chem. 2022 Jul 18. pii: S0021-9258(22)00725-6. [Epub ahead of print] 102283
      Knockout of the transcription factor X-box binding protein (XBP1) is known to decrease liver glucose production and lipogenesis. However, whether insulin can regulate gluconeogenesis and lipogenesis through XBP1 and how insulin activates the inositol-requiring enzyme (IRE1)-XBP1 ER stress pathway remain unexplored. Here we report that in the fed state, insulin-activated kinase AKT directly phosphorylates IRE1 at S724, which in turn mediates the splicing of XBP1u mRNA, thus favoring the generation of the spliced form, XBP1s, in the liver of mice. Subsequently, XBP1s stimulates the expression of lipogenic genes and upregulates liver lipogenesis as previously reported. Intriguingly, we find that fasting leads to an increase in XBP1u along with a drastic decrease in XBP1s in the liver of mice, and XBP1u, not XBP1s, significantly increases PKA-stimulated CRE reporter activity in cultured hepatocytes. Furthermore, we demonstrate overexpression of XBP1u significantly increases cAMP-stimulated expression of rate-limiting gluconeogenic genes, G6pc and Pck1, and glucose production in primary hepatocytes. Re-expression of XBP1u in the liver of mice with XBP1 depletion significantly increases fasted blood glucose levels and gluconeogenic gene expression. These data support an important role of XBP1u in upregulating gluconeogenesis in the fasted state. Taken together, we reveal that insulin signaling via AKT controls the expression of XBP1 isoforms, and that XBP1u and XBP1s function in different nutritional states to regulate liver gluconeogenesis and lipogenesis, respectively.
    Keywords:  AKT; IRE1; XBP1; insulin; liver glucose production; triglyceride
  10. Science. 2022 Jul 14. eabf4062
      Individual cells take decisions that are adapted to their internal state and surroundings, but how cells can reliably do this remains unclear. Using multiplexed quantification of signaling responses and markers of the cellular state, we find that signaling nodes in a network display adaptive information processing, which leads to heterogeneous growth factor responses and enables nodes to capture partially non-redundant information about the cellular state. Collectively, as a multimodal percept, this gives individual cells a large information processing capacity to accurately place growth factor concentration within the context of their cellular state and make cellular state-dependent decisions. We propose that heterogeneity and complexity in signaling networks have co-evolved to enable specific and context-aware cellular decision making in a multicellular setting.
  11. iScience. 2022 Aug 19. 25(8): 104688
      Metabolic perturbations can affect gene expression, for instance to rewire metabolism. While numerous efforts have measured gene expression in response to individual metabolic perturbations, methods that determine all metabolic perturbations that affect the expression for a given gene or set of genes have not been available. Here, we use a gene-centered approach to derive a first-pass metabolic regulatory network for Caenorhabditis elegans by performing RNAi of more than 1,400 metabolic genes with a set of 19 promoter reporter strains that express a fluorescent protein in the animal's intestine. We find that metabolic perturbations generally increase promoter activity, which contrasts with transcription factor (TF) RNAi, which tends to repress promoter activity. We identify several TFs that modulate promoter activity in response to perturbations of the electron transport chain and explore complex genetic interactions among metabolic pathways. This work provides a blueprint for a systems-level understanding of how metabolism affects gene expression.
    Keywords:  Experimental models in systems biology; Molecular biology; Systems biology
  12. Proc Natl Acad Sci U S A. 2022 Jul 19. 119(29): e2204527119
      Mice with insulin receptor (IR)-deficient astrocytes (GFAP-IR knockout [KO] mice) show blunted responses to insulin and reduced brain glucose uptake, whereas IR-deficient astrocytes show disturbed mitochondrial responses to glucose. While exploring the functional impact of disturbed mitochondrial function in astrocytes, we observed that GFAP-IR KO mice show uncoupling of brain blood flow with glucose uptake. Since IR-deficient astrocytes show higher levels of reactive oxidant species (ROS), this leads to stimulation of hypoxia-inducible factor-1α and, consequently, of the vascular endothelial growth factor angiogenic pathway. Indeed, GFAP-IR KO mice show disturbed brain vascularity and blood flow that is normalized by treatment with the antioxidant N-acetylcysteine (NAC). NAC ameliorated high ROS levels, normalized angiogenic signaling and mitochondrial function in IR-deficient astrocytes, and normalized neurovascular coupling in GFAP-IR KO mice. Our results indicate that by modulating glucose uptake and angiogenesis, insulin receptors in astrocytes participate in neurovascular coupling.
    Keywords:  astrocytes; insulin; neurovascular coupling
  13. JCI Insight. 2022 Jul 19. pii: e155899. [Epub ahead of print]
      Metastatic urothelial carcinoma is generally incurable with current systemic therapies. Chromatin modifiers are frequently mutated in bladder cancer, with ARID1A-inactivating mutations present in about 20% of tumors. EZH2, a histone methyltransferase, acts as an oncogene that functionally opposes ARID1A. In addition, PI3K signaling is activated in more than 20% of bladder cancers. Using a combination of in vitro and in vivo data, including patient derived xenografts, we show that ARID1A-mutant tumors are more sensitive to EZH2 inhibition than ARID1A-wild type tumors. Mechanistic studies reveal that: 1) ARID1A deficiency results in a dependency on PI3K/AKT/mTOR signaling via novel upregulation of a non-canonical PI3K regulatory subunit, PIK3R3, and downregulation of MAPK signaling, and: 2) EZH2 inhibitor sensitivity is due to upregulation of PIK3IP1, a protein inhibitor of PI3K signaling. We show for the first time that PIK3IP1 inhibits PI3K signaling by inducing proteasomal degradation of PIK3R3. Further, ARID1A deficient bladder cancer is sensitive to combination therapies with EZH2 and PI3K inhibitors, in a synergistic manner. Thus, our studies suggest that bladder cancers with ARID1A mutations can be treated with inhibitors of EZH2 and/or PI3K, and reveal mechanistic insights into the role of non-canonical PI3K constituents in bladder cancer biology.
    Keywords:  Oncology; Urology
  14. Sci Rep. 2022 Jul 19. 12(1): 12311
      Dendritic cells (DCs) play pivotal roles in initiating and shaping both innate and adaptive immune responses. The spatiotemporal expression of transcription factor networks and activation of specific signal transduction pathways determine the specification, distribution and differentiation of DC subsets. Even though pioneering studies have established indispensable roles for specific catalytic subunits (p110δ and p110γ) in immune cells, functions of the regulatory subunits, particularly of Class I PI3K, within the hematopoietic system remain incompletely understood. In the study presented here, we deleted the key regulatory subunits-p85α and p85β of the Class IA PI3K in hematopoietic cells and studied its impact on DC differentiation. Our studies identify that a deficiency of p85 causes increased differentiation of conventional DC (cDC) 2 and plasmacytoid DC (pDC) subsets in the spleen. On the other hand, DC numbers in the bone marrow (BM), thymus and lymph nodes were decreased in p85 mutant mice. Analysis of DC-specific progenitors and precursors indicated increased numbers in the BM and spleen of p85 deficient mice. In-vitro differentiation studies demonstrated augmented DC-differentiation capacities of p85 deficient BM cells in the presence of GM-CSF and Flt3L. BM chimera studies established that p85 deficiency affects DC development through cell intrinsic mechanisms. Molecular studies revealed increased proliferation of DCs and common DC progenitors (CDPs) in the absence of p85 and altered signal transduction pathways in p85 mutant DC subsets in response to Flt3L. In essence, data presented here, for the first time, unequivocally establish that the P85α subunit of class IA PI3Ks has an indispensable role in the development and maintenance of DCs.
  15. Cell. 2022 Jul 21. pii: S0092-8674(22)00844-3. [Epub ahead of print]185(15): 2756-2769
      For decades, insight into fundamental principles of human biology and disease has been obtained primarily by experiments in animal models. While this has allowed researchers to understand many human biological processes in great detail, some developmental and disease mechanisms have proven difficult to study due to inherent species differences. The advent of organoid technology more than 10 years ago has established laboratory-grown organ tissues as an additional model system to recapitulate human-specific aspects of biology. The use of human 3D organoids, as well as other advances in single-cell technologies, has revealed unprecedented insights into human biology and disease mechanisms, especially those that distinguish humans from other species. This review highlights novel advances in organoid biology with a focus on how organoid technology has generated a better understanding of human-specific processes in development and disease.
    Keywords:  disease; in vitro model; organoid; stem cells
  16. Methods Mol Biol. 2022 ;2535 141-156
      CRISPR-Cas9 genome editing provides a means for simple and scalable production of gene knockouts in mammalian cell lines. The development of guide RNA (gRNA) libraries targeting tens of thousands of genes has allowed researchers to produce pools of cells, each containing a single gene knockout for use in genetic screens. In addition to assessing the effect of gene knockout on cell proliferation, CRISPR-Cas9 genetic screens can be used to assess gene-drug interactions. Here, we outline a protocol for performing positive and negative selection genome-wide CRISPR-Cas9 screens for identifying gene knockouts that cause drug resistance and hypersensitivity. This protocol is designed for the use of the TKOv3 library in human cell lines, but can be readily adapted for different libraries.
    Keywords:  CRISPR-Cas9; Chemogenomics; Drug resistance; Functional genomics
  17. ACS Omega. 2022 Jul 12. 7(27): 23479-23486
      In cancer, the mechanistic/mammalian target of rapamycin complex-1 (mTORC1) is hyperactivated to promote survival under adverse conditions. The kinase activity of mTORC1 is activated by small-GTPase RHEB-GTP. Therefore, a new modality to inhibit mTORC1 activity has emerged, through intercepting RHEB. However, due to the relatively large contact area involved in the interaction between RHEB and mTORC1, facilitating this inhibition through small molecules has been challenging. Here, we report the development of a peptide that can inhibit the RHEB-mTORC1 interaction. The peptide, P1_WT, was designed based on the α-helix (aa 101-115) of the N-heat domain of mTOR to interact with switch II of RHEB. P1_WT bound to RHEB (K D = 0.14 μM) and inhibited RHEB-mTORN-heat interaction (IC50 = 0.33 μM) in vitro. Consequently, P1_WT inhibited mTORC1 activity at a sub-micromolar level (IC50 ∼ 0.3 μM). P1_WT was predicted to be cell-permeable due to the rich content of arginine (23%), enhancing the intracellular translocation. These results show that P1_WT is a potential compound to further develop inhibitors for mTORC1 by intercepting RHEB from mTORC1.
  18. Elife. 2022 Jul 20. pii: e80609. [Epub ahead of print]11
      An automated platform for cell culture combines robotics and artificial intelligence to optimize cell culture protocols and reliably produce specific cell types that could be used for regenerative medicine treatments.
    Keywords:  bayesian optimization; computational biology; human; ips cell; labdroid; laboratory automation; regenerative medicine; retinal pigment epithelium; stem cells; systems biology