bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2021‒05‒30
twenty-one papers selected by
Ralitsa Radostinova Madsen
University College London Cancer Institute

  1. Nat Commun. 2021 May 25. 12(1): 3140
      INPP4B suppresses PI3K/AKT signaling by converting PI(3,4)P2 to PI(3)P and INPP4B inactivation is common in triple-negative breast cancer. Paradoxically, INPP4B is also a reported oncogene in other cancers. How these opposing INPP4B roles relate to PI3K regulation is unclear. We report PIK3CA-mutant ER+ breast cancers exhibit increased INPP4B mRNA and protein expression and INPP4B increased the proliferation and tumor growth of PIK3CA-mutant ER+ breast cancer cells, despite suppression of AKT signaling. We used integrated proteomics, transcriptomics and imaging to demonstrate INPP4B localized to late endosomes via interaction with Rab7, which increased endosomal PI3Kα-dependent PI(3,4)P2 to PI(3)P conversion, late endosome/lysosome number and cargo trafficking, resulting in enhanced GSK3β lysosomal degradation and activation of Wnt/β-catenin signaling. Mechanistically, Wnt inhibition or depletion of the PI(3)P-effector, Hrs, reduced INPP4B-mediated cell proliferation and tumor growth. Therefore, INPP4B facilitates PI3Kα crosstalk with Wnt signaling in ER+ breast cancer via PI(3,4)P2 to PI(3)P conversion on late endosomes, suggesting these tumors may be targeted with combined PI3K and Wnt/β-catenin therapies.
  2. Cell Mol Life Sci. 2021 May 27.
      Fatty acid synthase (FASN) participates in many fundamental biological processes, including energy storage and signal transduction, and is overexpressed in many cancer cells. We previously showed in a context of lipogenesis that FASN is protected from degradation by its interaction with O-GlcNAc transferase (OGT) in a nutrient-dependent manner. We and others also reported that OGT and O-GlcNAcylation up-regulate the PI3K/AKT/mTOR pathway that senses mitogenic signals and nutrient availability to drive cell cycle. Using biochemical and microscopy approaches, we show here that FASN co-localizes with OGT in the cytoplasm and, to a lesser extent, in the membrane fraction. This interaction occurs in a cell cycle-dependent manner, following the pattern of FASN expression. Moreover, we show that FASN expression depends on OGT upon serum stimulation. The level of FASN also correlates with the activation of the PI3K/AKT/mTOR pathway in hepatic cell lines, and in livers of obese mice and in a chronically activated insulin and mTOR signaling mouse model (PTEN-null mice). These results indicate that FASN is under a dual control of O-GlcNAcylation and mTOR pathways. In turn, blocking FASN with the small-molecule inhibitor C75 reduces both OGT and O-GlcNAcylation levels, and mTOR activation, highlighting a novel reciprocal regulation between these actors. In addition to the role of O-GlcNAcylation in tumorigenesis, our findings shed new light on how aberrant activity of FASN and mTOR signaling may promote the emergence of hepatic tumors.
    Keywords:  Cell proliferation; Ob/ob mice; Protein interactions; Proximity ligation assay; siRNA
  3. EMBO Rep. 2021 May 27. e53232
      Lowe syndrome is a rare, developmental disorder caused by mutations in the phosphatase, OCRL. A study in this issue of EMBO Reports shows that OCRL is required for microtubule nucleation and that mutations in this protein lead to an inability to activate mTORC1 signaling and consequent cell proliferation in the presence of nutrients. These defects are the result of impaired microtubule-dependent lysosomal trafficking to the cell periphery and are independent of OCRL phosphatase activity.
  4. Circulation. 2021 May 25.
      Background: The cellular diversity of the lung endothelium has not been systematically characterized in humans. Here, we provide a reference atlas of human lung endothelial cells (ECs) to facilitate a better understanding of the phenotypic diversity and composition of cells comprising the lung endothelium. Methods: We reprocessed human control single cell RNA sequencing (scRNAseq) data from six datasets. EC populations were characterized through iterative clustering with subsequent differential expression analysis. Marker genes were validated by fluorescent microscopy and in situ hybridization. scRNAseq of primary lung ECs cultured in-vitro was performed. The signaling network between different lung cell types was studied. For cross species analysis or disease relevance, we applied the same methods to scRNAseq data obtained from mouse lungs or from human lungs with pulmonary hypertension. Results: Six lung scRNAseq datasets were reanalyzed and annotated to identify over 15,000 vascular EC cells from 73 individuals. Differential expression analysis of EC revealed signatures corresponding to endothelial lineage, including pan-endothelial, pan-vascular and subpopulation-specific marker gene sets. Beyond the broad cellular categories of lymphatic, capillary, arterial and venous ECs, we found previously indistinguishable subpopulations: among venous EC, we identified two previously indistinguishable populations, pulmonary-venous ECs (COL15A1neg) localized to the lung parenchyma and systemic-venous ECs (COL15A1pos) localized to the airways and the visceral pleura; among capillary EC, we confirmed their subclassification into recently discovered aerocytes characterized by EDNRB, SOSTDC1 and TBX2 and general capillary EC. We confirmed that all six endothelial cell types, including the systemic-venous EC and aerocytes, are present in mice and identified endothelial marker genes conserved in humans and mice. Ligand-receptor connectome analysis revealed important homeostatic crosstalk of EC with other lung resident cell types. scRNAseq of commercially available primary lung ECs demonstrated a loss of their native lung phenotype in culture. scRNAseq revealed that the endothelial diversity is maintained in pulmonary hypertension. Our manuscript is accompanied by an online data mining tool ( Conclusions: Our integrated analysis provides the comprehensive and well-crafted reference atlas of lung endothelial cells in the normal lung and confirms and describes in detail previously unrecognized endothelial populations across a large number of humans and mice.
  5. Genet Med. 2021 May 26.
      PURPOSE: Somatic activating variants in the PI3K-AKT pathway cause vascular malformations with and without overgrowth. We previously reported an individual with capillary and lymphatic malformation harboring a pathogenic somatic variant in PIK3R1, which encodes three PI3K complex regulatory subunits. Here, we investigate PIK3R1 in a large cohort with vascular anomalies and identify an additional 16 individuals with somatic mosaic variants in PIK3R1.METHODS: Affected tissue from individuals with vascular lesions and overgrowth recruited from a multisite collaborative network was studied. Next-generation sequencing targeting coding regions of cell-signaling and cancer-associated genes was performed followed by assessment of variant pathogenicity.
    RESULTS: The phenotypic and variant spectrum associated with somatic variation in PIK3R1 is reported herein. Variants occurred in the inter-SH2 or N-terminal SH2 domains of all three PIK3R1 protein products. Phenotypic features overlapped those of the PIK3CA-related overgrowth spectrum (PROS). These overlapping features included mixed vascular malformations, sandal toe gap deformity with macrodactyly, lymphatic malformations, venous ectasias, and overgrowth of soft tissue or bone.
    CONCLUSION: Somatic PIK3R1 variants sharing attributes with cancer-associated variants cause complex vascular malformations and overgrowth. The PIK3R1-associated phenotypic spectrum overlaps with PROS. These data extend understanding of the diverse phenotypic spectrum attributable to genetic variation in the PI3K-AKT pathway.
  6. Nat Commun. 2021 May 24. 12(1): 3055
      Triple negative breast cancer (TNBC) patients exhibit poor survival outcomes and lack effective targeted therapies. Using unbiased in vivo genome-wide CRISPR screening, we interrogated cancer vulnerabilities in TNBC and identified an interplay between oncogenic and tumor suppressor pathways. This study reveals tumor regulatory functions for essential components of the mTOR and Hippo pathways in TNBC. Using in vitro drug matrix synergy models and in vivo patient-derived xenografts, we further establish the therapeutic relevance of our findings and show that pharmacological inhibition of mTORC1/2 and oncoprotein YAP efficiently reduces tumorigenesis in TNBC. At the molecular level, we find that while verteporfin-induced YAP inhibition leads to apoptosis, torin1-mediated mTORC1/2 inhibition promotes macropinocytosis. Torin1-induced macropinocytosis further facilitates verteporfin uptake, thereby greatly enhancing its pro-apoptotic effects in cancer cells. Overall, our study underscores the power and robustness of in vivo CRISPR genome-wide screens in identifying clinically relevant and innovative therapeutic modalities in cancer.
  7. Nat Metab. 2021 May;3(5): 714-727
      Single-cell motility is spatially heterogeneous and driven by metabolic energy. Directly linking cell motility to cell metabolism is technically challenging but biologically important. Here, we use single-cell metabolic imaging to measure glycolysis in individual endothelial cells with genetically encoded biosensors capable of deciphering metabolic heterogeneity at subcellular resolution. We show that cellular glycolysis fuels endothelial activation, migration and contraction and that sites of high lactate production colocalize with active cytoskeletal remodelling within an endothelial cell. Mechanistically, RhoA induces endothelial glycolysis for the phosphorylation of cofilin and myosin light chain in order to reorganize the cytoskeleton and thus control cell motility; RhoA activation triggers a glycolytic burst through the translocation of the glucose transporter SLC2A3/GLUT3 to fuel the cellular contractile machinery, as demonstrated across multiple endothelial cell types. Our data indicate that Rho-GTPase signalling coordinates energy metabolism with cytoskeleton remodelling to regulate endothelial cell motility.
  8. Brain. 2021 May 28. pii: awab173. [Epub ahead of print]
      Phosphatase and tensin homolog (PTEN) regulates cell growth and survival through inhibition of the mammalian target of rapamycin (MTOR) signaling pathway. Germline genetic variation of PTEN is associated with autism, macrocephaly, and PTEN hamartoma tumor syndromes (PHTS). The effect of developmental PTEN somatic mutations on nervous system phenotypes is not well understood, although brain somatic mosaicism of MTOR pathway genes is an emerging cause of cortical dysplasia and epilepsy in the pediatric population. Here we report two somatic variants of PTEN affecting a single patient presenting with intractable epilepsy and hemimegalencephaly that varied in clinical severity throughout the left cerebral hemisphere. High-throughput sequencing analysis of affected brain tissue identified two somatic variants in PTEN. The first variant was present in multiple cell lineages throughout the entire hemisphere and associated with mild cerebral overgrowth. The second variant was restricted to posterior brain regions and affected the opposite PTEN allele, resulting in a segmental region of more severe malformation, and the only neurons in which it was found by single-nuclei RNA-seq had a unique disease-related expression profile. This study reveals brain mosaicism of PTEN as a disease mechanism of hemimegalencephaly and furthermore demonstrates the varying effects of single- or bi-allelic disruption of PTEN on cortical phenotypes.
    Keywords:  brain development; epilepsy; hemimegalencephaly (HME); somatic mosaicism
  9. Genome Biol. 2021 May 25. 22(1): 163
      A pressing challenge in single-cell transcriptomics is to benchmark experimental protocols and computational methods. A solution is to use computational simulators, but existing simulators cannot simultaneously achieve three goals: preserving genes, capturing gene correlations, and generating any number of cells with varying sequencing depths. To fill this gap, we propose scDesign2, a transparent simulator that achieves all three goals and generates high-fidelity synthetic data for multiple single-cell gene expression count-based technologies. In particular, scDesign2 is advantageous in its transparent use of probabilistic models and its ability to capture gene correlations via copulas.
  10. Nat Commun. 2021 May 24. 12(1): 3059
      Non-alcoholic fatty liver disease (NAFLD) has become the most prevalent chronic liver disease in the world, however, no drug treatment has been approved for this disease. Thus, it is urgent to find effective therapeutic targets for clinical intervention. In this study, we find that liver-specific knockout of PPDPF (PPDPF-LKO) leads to spontaneous fatty liver formation in a mouse model at 32 weeks of age on chow diets, which is enhanced by HFD. Mechanistic study reveals that PPDPF negatively regulates mTORC1-S6K-SREBP1 signaling. PPDPF interferes with the interaction between Raptor and CUL4B-DDB1, an E3 ligase complex, which prevents ubiquitination and activation of Raptor. Accordingly, liver-specific PPDPF overexpression effectively inhibits HFD-induced mTOR signaling activation and hepatic steatosis in mice. These results suggest that PPDPF is a regulator of mTORC1 signaling in lipid metabolism, and may be a potential therapeutic candidate for NAFLD.
  11. Endocrinology. 2021 May 25. pii: bqab104. [Epub ahead of print]
      Endothelial insulin receptors (Insr) promote sprouting angiogenesis, although the underpinning cellular and molecular mechanisms are unknown. Comparing mice with whole body insulin receptor haploinsufficiency (Insr +/-) against littermate controls, we found impaired limb perfusion and muscle capillary density after inducing hind-limb ischemia; this was in spite of increased expression of the pro-angiogenic growth factor Vegfa. Insr +/- neonatal retinas exhibited reduced tip cell number and branching complexity during developmental angiogenesis, which was also found in separate studies of mice with endothelium-restricted Insr haploinsufficiency. Functional responses to VEGF-A, including in vitro angiogenesis, were also impaired in aortic rings and pulmonary endothelial cells from Insr +/- mice. Human umbilical vein endothelial cells (HUVEC) with shRNA-mediated knockdown of Insr also demonstrated impaired functional angiogenic responses to VEGF-A. VEGF-A signaling to Akt and eNOS was intact, but downstream signaling to ERK1/2 was impaired, as was VEGF receptor-2 (VEGFR-2) internalization, which is required specifically for signaling to ERK1/2. Hence, endothelial insulin receptors facilitate the functional response to VEGF-A during angiogenic sprouting and are required for appropriate signal transduction from VEGFR-2 to ERK1/2.
    Keywords:  Angiogenesis; ERK; Endothelial; Insulin; VEGF; Vascular
  12. Front Mol Biosci. 2021 ;8 648663
      Dysregulation of phosphoinositide 3-kinase (PI3K) signaling is highly implicated in tumorigenesis, disease progression, and the development of resistance to the current standard of care treatments in breast cancer patients. This review discusses the role of PI3K pathway in breast cancer and evaluates the clinical development of PI3K inhibitors in both early and metastatic breast cancer settings. Further, this review examines the evidence for the potential synergistic benefit for the combination treatment of PI3K inhibition and immunotherapy in breast cancer treatment.
    Keywords:  MDSC; PI3K inhibition; breast cancer; immune checkpoint inhibitors; tumor immune microenvironment
  13. Trends Cancer. 2021 May 21. pii: S2405-8033(21)00101-1. [Epub ahead of print]
      Cancer is the dysregulated proliferation of cells caused by acquired mutations in key driver genes. The most frequently mutated driver genes promote tumorigenesis in various organisms, cell types, and genetic backgrounds. However, recent cancer genomics studies also point to the existence of context-dependent driver gene functions, where specific mutations occur predominately or even exclusively in certain tumor types or genetic backgrounds. Here, we review examples of co-occurring and mutually exclusive driver gene mutation patterns across cancer genomes and discuss their underlying biology. While co-occurring driver genes typically activate collaborating oncogenic pathways, we identify two distinct biological categories of incompatibilities among the mutually exclusive driver genes depending on whether the mutated drivers trigger the same or divergent tumorigenic pathways. Finally, we discuss possible therapeutic avenues emerging from the study of incompatible driver gene mutations.
    Keywords:  cancer driver mutations; co-occurrence; driver genes antagonism; genomic patterns; mutual exclusivity; pathway redundancy and divergence; synergy; synthetic essentiality; synthetic lethality
  14. EMBO Mol Med. 2021 May 25. e13502
      Pancreatic ductal adenocarcinoma (PDAC) patients frequently suffer from undetected micro-metastatic disease. This clinical situation would greatly benefit from additional investigation. Therefore, we set out to identify key signalling events that drive metastatic evolution from the pancreas. We searched for a gene signature that discriminate localised PDAC from confirmed metastatic PDAC and devised a preclinical protocol using circulating cell-free DNA (cfDNA) as an early biomarker of micro-metastatic disease to validate the identification of key signalling events. An unbiased approach identified, amongst actionable markers of disease progression, the PI3K pathway and a distinctive PI3Kα activation signature as predictive of PDAC aggressiveness and prognosis. Pharmacological or tumour-restricted genetic PI3Kα-selective inhibition prevented macro-metastatic evolution by hindering tumoural cell migratory behaviour independently of genetic alterations. We found that PI3Kα inhibition altered the quantity and the species composition of the produced lipid second messenger PIP3 , with a selective decrease of C36:2 PI-3,4,5-P3 . Tumoural PI3Kα inactivation prevented the accumulation of pro-tumoural CD206-positive macrophages in the tumour-adjacent tissue. Tumour cell-intrinsic PI3Kα promotes pro-metastatic features that could be pharmacologically targeted to delay macro-metastatic evolution.
    Keywords:  PI3K isoforms; pancreatic cancer; phosphoinositide; targeted therapy; tumour-stroma dialog
  15. Mol Cell Oncol. 2021 Mar 25. 8(3): 1902250
      The rate-limiting enzyme of serine biosynthesis, 3-phosphoglycerate dehydrogenase (PHGDH), contributes to rapid growth and proliferation when it is overexpressed in cancer. We recently described the metabolic adaptations that occur upon PHGDH inhibition in osteosarcoma. PHGDH inhibition causes metabolite accumulation that activates the mechanistic target of rapamycin (mTOR) signaling, sensitizing osteosarcoma to non-rapalog mTOR inhibition.
    Keywords:  PHGDH; mTORC1; methotrexate; osteosarcoma; perhexiline; serine
  16. NPJ Breast Cancer. 2021 May 28. 7(1): 66
      Breast cancer is the most commonly diagnosed cancer in the USA. Although advances in treatment over the past several decades have significantly improved the outlook for this disease, most women who are diagnosed with estrogen receptor positive disease remain at risk of metastatic relapse for the remainder of their life. The cellular source of late relapse in these patients is thought to be disseminated tumor cells that reactivate after a long period of dormancy. The biology of these dormant cells and their natural history over a patient's lifetime is largely unclear. We posit that research on tumor dormancy has been significantly limited by the lack of clinically relevant models. This review will discuss existing dormancy models, gaps in biological understanding, and propose criteria for future models to enhance their clinical relevance.
  17. JCI Insight. 2021 May 25. pii: 149271. [Epub ahead of print]
      Fetal growth restriction, or low birthweight is a strong determinant for eventual obesity and Type 2 diabetes. Clinical studies suggest placental mechanistic target of rapamycin (mTOR) signaling regulate fetal birthweight and the metabolic health trajectory of the offspring. In the current study, we used genetic model with loss of placental mTOR function (mTORKOPlacenta) to test the direct role of mTOR signaling on birthweight and the metabolic health in the adult offspring. mTORKOPlacenta animals displayed reduced placental area and total weight, as well as fetal bodyweight at embryonic day (e) 17.5. Birthweight and serum insulin levels were reduced; however, β-cell mass was normal in mTORKOPlacenta newborns. Adult mTORKOPlacenta offspring, under a metabolic high-fat challenge, displayed exacerbated obesity and metabolic dysfunction compared to littermate controls. Subsequently, we tested whether enhancing placental mTOR complex 1 (mTORC1) signaling, via genetic ablation of TSC2, in utero would improve glucose homeostasis in the offspring. Indeed, increased placental mTORC1 conferred protection from a diet-induced obesity in the offspring. In conclusion, placental mTORC1 serves as a mechanistic link between placental function and programming of obesity and insulin resistance in the adult offspring.
    Keywords:  Diabetes; Endocrinology; Islet cells; Metabolism; Obesity
  18. Nat Commun. 2021 May 24. 12(1): 3042
      Controlling off-target editing activity is one of the central challenges in making CRISPR technology accurate and applicable in medical practice. Current algorithms for analyzing off-target activity do not provide statistical quantification, are not sufficiently sensitive in separating signal from noise in experiments with low editing rates, and do not address the detection of translocations. Here we present CRISPECTOR, a software tool that supports the detection and quantification of on- and off-target genome-editing activity from NGS data using paired treatment/control CRISPR experiments. In particular, CRISPECTOR facilitates the statistical analysis of NGS data from multiplex-PCR comparative experiments to detect and quantify adverse translocation events. We validate the observed results and show independent evidence of the occurrence of translocations in human cell lines, after genome editing. Our methodology is based on a statistical model comparison approach leading to better false-negative rates in sites with weak yet significant off-target activity.
  19. Chem Commun (Camb). 2021 May 24.
      Optochemical tools that can modulate the activity of the target protein provide an opportunity for studying and regulating the related biological processes. Here we present a DNA-based nongenetic optochemical tool that can control the dynamics of growth factor signaling. This photo-caged mimicry of growth factor can be a promising tool for elucidating a linkage between the dynamics of signaling and the resulting biological outcomes, as well as for manipulating cellular functions and the fate of living cells.
  20. BMC Bioinformatics. 2021 May 24. 22(1): 262
      BACKGROUND: Biological tissues consist of heterogenous populations of cells. Because gene expression patterns from bulk tissue samples reflect the contributions from all cells in the tissue, understanding the contribution of individual cell types to the overall gene expression in the tissue is fundamentally important. We recently developed a computational method, CDSeq, that can simultaneously estimate both sample-specific cell-type proportions and cell-type-specific gene expression profiles using only bulk RNA-Seq counts from multiple samples. Here we present an R implementation of CDSeq (CDSeqR) with significant performance improvement over the original implementation in MATLAB and an added new function to aid cell type annotation. The R package would be of interest for the broader R community.RESULT: We developed a novel strategy to substantially improve computational efficiency in both speed and memory usage. In addition, we designed and implemented a new function for annotating the CDSeq estimated cell types using single-cell RNA sequencing (scRNA-seq) data. This function allows users to readily interpret and visualize the CDSeq estimated cell types. In addition, this new function further allows the users to annotate CDSeq-estimated cell types using marker genes. We carried out additional validations of the CDSeqR software using synthetic, real cell mixtures, and real bulk RNA-seq data from the Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression (GTEx) project.
    CONCLUSIONS: The existing bulk RNA-seq repositories, such as TCGA and GTEx, provide enormous resources for better understanding changes in transcriptomics and human diseases. They are also potentially useful for studying cell-cell interactions in the tissue microenvironment. Bulk level analyses neglect tissue heterogeneity, however, and hinder investigation of a cell-type-specific expression. The CDSeqR package may aid in silico dissection of bulk expression data, enabling researchers to recover cell-type-specific information.
    Keywords:  CDSeq; Deconvolution; Gene expression; Tissue heterogeneity
  21. Cell Rep. 2021 May 25. pii: S2211-1247(21)00499-X. [Epub ahead of print]35(8): 109157
      Increasing evidence suggests that the reactivation of initially inhibited signaling pathways causes drug resistance. Here, we analyze how network topologies affect signaling responses to drug treatment. Network-dependent drug resistance is commonly attributed to negative and positive feedback loops. However, feedback loops by themselves cannot completely reactivate steady-state signaling. Newly synthesized negative feedback regulators can induce a transient overshoot but cannot fully restore output signaling. Complete signaling reactivation can only occur when at least two routes, an activating and inhibitory, connect an inhibited upstream protein to a downstream output. Irrespective of the network topology, drug-induced overexpression or increase in target dimerization can restore or even paradoxically increase downstream pathway activity. Kinase dimerization cooperates with inhibitor-mediated alleviation of negative feedback. Our findings inform drug development by considering network context and optimizing the design drug combinations. As an example, we predict and experimentally confirm specific combinations of RAF inhibitors that block mutant NRAS signaling.
    Keywords:  MAPK pathways; RAF inhibitors; drug resistance; drug synergy; kinase inhibitors; kinetic models; modular response analysis; oncogenic RAS; signaling networks; targeted therapy