bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2023‒05‒21
fifteen papers selected by
Ralitsa Radostinova Madsen

  1. Acta Mater Med. 2022 ;1(2): 193-196
      The classical phosphatidylinositol 3-kinases (PI3Ks) are heterodimers of p110 and p85. PIK3CA, the gene encoding the catalytic p110α subunit, is one of the most frequently mutated oncogenes in human cancers with hot spot mutations occurring in the helical domain or in the kinase domain. Tumors with these two types of PIK3CA mutations show overlapping yet distinct phenotypes; however, the underlying mechanisms remain unclear. In a recent publication [1], Hao et al revealed exciting findings about the PI3K p85β regulatory subunit in promoting PIK3CA helical domain mutation-driven cancer progression. The authors found that p85β disassociated from the PI3K complex and translocated into the nucleus only in cancer cells harboring PIK3CA helical domain mutations. Disrupting nuclear localization of p85β suppressed mouse tumor growth of cancer cells with PIK3CA helical domain mutation. Mechanistically, they elegantly showed that nuclear p85β recruited the deubiquitinase USP7 to stabilize the histone methyltransferases EZH1/2, leading to enhanced H3K27 trimethylation and gene transcription. Combining an EZH inhibitor with a PI3K inhibitor specifically resulted in regression of mouse xenograft tumors with PIK3CA helical domain mutations. These findings illustrate a previously uncharacterized function of p85β in tumor development and suggest an effective approach to target tumors with PIK3CA helical mutations.
    Keywords:  PI3K; cancer mutation; colorectal cancer; nuclear translocation; p110; p85
  2. Stem Cell Reports. 2023 May 09. pii: S2213-6711(23)00142-X. [Epub ahead of print]
      Signaling is central in cell fate regulation, and relevant information is encoded in its activity over time (i.e., dynamics). However, simultaneous dynamics quantification of several pathways in single mammalian stem cells has not yet been accomplished. Here we generate mouse embryonic stem cell (ESC) lines simultaneously expressing fluorescent reporters for ERK, AKT, and STAT3 signaling activity, which all control pluripotency. We quantify their single-cell dynamics combinations in response to different self-renewal stimuli and find striking heterogeneity for all pathways, some dependent on cell cycle but not pluripotency states, even in ESC populations currently assumed to be highly homogeneous. Pathways are mostly independently regulated, but some context-dependent correlations exist. These quantifications reveal surprising single-cell heterogeneity in the important cell fate control layer of signaling dynamics combinations and raise fundamental questions about the role of signaling in (stem) cell fate control.
    Keywords:  AKT; ERK; ESC; STAT3; dynamics; heterogeneity; signaling; time-lapse
  3. Dev Cell. 2023 May 13. pii: S1534-5807(23)00183-1. [Epub ahead of print]
      ERK and AKT signaling control pluripotent cell self-renewal versus differentiation. ERK pathway activity over time (i.e., dynamics) is heterogeneous between individual pluripotent cells, even in response to the same stimuli. To analyze potential functions of ERK and AKT dynamics in controlling mouse embryonic stem cell (ESC) fates, we developed ESC lines and experimental pipelines for the simultaneous long-term manipulation and quantification of ERK or AKT dynamics and cell fates. We show that ERK activity duration or amplitude or the type of ERK dynamics (e.g., transient, sustained, or oscillatory) alone does not influence exit from pluripotency, but the sum of activity over time does. Interestingly, cells retain memory of previous ERK pulses, with duration of memory retention dependent on duration of previous pulse length. FGF receptor/AKT dynamics counteract ERK-induced pluripotency exit. These findings improve our understanding of how cells integrate dynamics from multiple signaling pathways and translate them into cell fate cues.
    Keywords:  Akt; Erk; cell fate decisions; dynamics; mouse embryonic stem cells; optogenetics; quantitative imaging; signaling; time-lapse imaging
  4. bioRxiv. 2023 May 01. pii: 2023.05.01.538969. [Epub ahead of print]
      The class 1A phosphoinositide 3-kinase (PI3K) beta (PI3Kβ) is functionally unique in the ability to integrate signals derived from receptor tyrosine kinases (RTKs), heterotrimeric guanine nucleotide-binding protein (G-protein)-coupled receptors (GPCRs), and Rho-family GTPases. The mechanism by which PI3Kβ prioritizes interactions with various membrane tethered signaling inputs, however, remains unclear. Previous experiments have not been able to elucidate whether interactions with membrane-tethered proteins primarily control PI3Kβ localization versus directly modulate lipid kinase activity. To address this gap in our understanding of PI3Kβ regulation, we established an assay to directly visualize and decipher how three binding interactions regulate PI3Kβ when presented to the kinase in a biologically relevant configuration on supported lipid bilayers. Using single molecule Total Internal Reflection Fluorescence (TIRF) Microscopy, we determined the mechanism controlling membrane localization of PI3Kβ, prioritization of signaling inputs, and lipid kinase activation. We find that auto-inhibited PI3Kβ must first cooperatively engage a single RTK-derived tyrosine phosphorylated (pY) peptide before it can engage either GβGγ or Rac1(GTP). Although pY peptides strongly localize PI3Kβ to membranes, they only modestly stimulate lipid kinase activity. In the presence of either pY/GβGγ or pY/Rac1(GTP), PI3Kβ activity is dramatically enhanced beyond what can be explained by the increase in membrane avidity for these complexes. Instead, PI3Kβ is synergistically activated by pY/GβGγ and pY/Rac1(GTP) through a mechanism of allosteric regulation.
  5. Pediatr Blood Cancer. 2023 May 17. e30419
      Complex lymphatic anomalies are debilitating conditions characterized by aberrant development of the lymphatic vasculature (lymphangiogenesis). Diagnosis is typically made by history, examination, radiology, and histologic findings. However, there is significant overlap between conditions, making accurate diagnosis difficult. Recently, genetic analysis has been offered as an additional diagnostic modality. Here, we describe four cases of complex lymphatic anomalies, all with PIK3CA variants but with varying clinical phenotypes. Identification of PIK3CA resulted in transition to a targeted inhibitor, alpelisib. These cases highlight the genetic overlap between phenotypically diverse lymphatic anomalies.
    Keywords:  PIK3CA; central conducting lymphatic anomaly; complex lymphatic anomalies; generalized lymphatic anomaly; kaposiform lymphangiomatosis
  6. Cell Signal. 2023 May 17. pii: S0898-6568(23)00134-1. [Epub ahead of print] 110720
      Current therapeutic approaches for colorectal cancer (CRC) focus on the suppression of oncogenic kinase signaling. Here, we test the hypothesis that targeted hyperactivation of the PI3K/AKT-signaling may lead to trigger CRC cell death. Recently we found that hematopoietic SHIP1 is ectopically expressed in CRC cells. Here we show that SHIP1 is more strongly expressed in metastatic cells than in the primary cancer cells, which allows for an increase in AKT signaling in metastatic cells, giving them an advantage from an evolutionary point of view. Mechanistically, the increased SHIP1 expression reduces the activation of the PI3K/ AKT signaling to a value that is below the threshold that leads to cell death. This mechanism gives the cell a selection advantage. We show that genetic hyperactivation of PI3K/AKT-signaling or blocking the activity of the inhibitory phosphatase SHIP1, induces acute cell death in CRC cells, because of excessive accumulation of reactive oxygen species. Our results demonstrate that CRC cells critically depend on mechanisms to fine-tune PI3K/AKT activity and show SHIP1 inhibition as an unexpectedly promising concept for CRC therapy.
    Keywords:  Colorectal cancer; Inositol 5-phosphatase; Metastasis; PI3K/AKT-signaling; ROS; SHIP1; Targeted hyperactivation
  7. bioRxiv. 2023 May 05. pii: 2023.05.04.539492. [Epub ahead of print]
      The conversion of PIP2 to PIP3 by phosphoinositide 3-kinase γ (PI3Kγ) is a critical step in neutrophil chemotaxis and is essential for metastasis in many types of cancer. PI3Kγ is activated via directed interaction with Gβγ heterodimers released from cell-surface G protein-coupled receptors (GPCRs) responding to extracellular signals. To resolve how Gβγ activates PI3Kγ, we determined cryo-EM reconstructions of PI3Kγ-Gβγ complexes in the presence of various substrates/analogs, revealing two distinct Gβγ binding sites, one on the p110γ helical domain and one on the C-terminal domain of the p101 subunit. Comparison of these complexes with structures of PI3Kγ alone demonstrates conformational changes in the kinase domain upon Gβγ binding similar to those induced by Ras·GTP. Assays of variants perturbing the two Gβγ binding sites and interdomain contacts that change upon Gβγ binding suggest that Gβγ not only recruits the enzyme to membranes but also allosterically controls activity via both sites. Studies in a zebrafish model examining neutrophil migration are consistent with these results. These findings set the stage for future detailed investigation of Gβγ-mediated activation mechanisms in this enzyme family and will aid in developing drugs selective for PI3Kγ.
  8. Methods Mol Biol. 2023 ;2660 137-148
      Mass spectrometry (MS) is an important tool for biological studies because it is capable of interrogating a diversity of biomolecules (proteins, drugs, metabolites) not captured via alternate genomic platforms. Unfortunately, downstream data analysis becomes complicated when attempting to evaluate and integrate measurements of different molecular classes and requires the aggregation of expertise from different relevant disciplines. This complexity represents a significant bottleneck that limits the routine deployment of MS-based multi-omic methods, despite the unmatched biological and functional insight the data can provide. To address this unmet need, our group introduced Omics Notebook as an open-source framework for facilitating exploratory analysis, reporting and integrating MS-based multi-omic data in a way that is automated, reproducible and customizable. By deploying this pipeline, we have devised a framework for researchers to more rapidly identify functional patterns across complex data types and focus on statistically significant and biologically interesting aspects of their multi-omic profiling experiments. This chapter aims to describe a protocol which leverages our publicly accessible tools to analyze and integrate data from high-throughput proteomics and metabolomics experiments and produce reports that will facilitate more impactful research, cross-institutional collaborations, and wider data dissemination.
    Keywords:  Automated pipeline; Biological functional modules; Graphical user interface (GUI); Mass spectrometry; Metabolomics; Multi-omics data integration; Omics Notebook R package; Omics Notebook interface; Pathway enrichment; Principal component analysis (PCA); Proteomics; Proteomics search engines; Systems biology
  9. Mod Pathol. 2023 May 10. pii: S0893-3952(23)00118-7. [Epub ahead of print] 100213
      Gestational endometrium can demonstrate a spectrum of atypical but benign changes. One such lesion is Localized Endometrial Proliferation of Pregnancy (LEPP), first described in a series of 11 cases. To understand its biologic and clinical importance, we explore the pathologic, immunophenotypic and molecular features of this entity. Nine cases of LEPP identified in a 15-year period were retrieved from departmental archives and reviewed. Immunohistochemistry and next generation sequencing using a comprehensive 446-gene panel were performed when material was available. Eight cases were identified in curettage specimens performed after first-trimester pregnancy loss, and one in the basal plate of a mature placenta. Mean patient age was 35 (range 27-41) years. Mean lesion size was 6.3 (range 2-12) mm. Architectural patterns, often coexisting in the same case, included cribriform (n=7), solid (n=5), villoglandular (n=2), papillary (n=2) and micropapillary (n=1). Cytologic atypia was mild in 7 cases and moderate in two. Mitotic activity was low (up to 3 per 2.4 mm2). All lesions were associated with neutrophils. Background Arias-Stella phenomenon was present in 4 cases. Immunohistochemistry was performed in 7 LEPP, all of which demonstrated wildtype p53, retained MSH6 and PMS2, membranous beta-catenin, and positive ER (mean 71%) and PR (mean 74%). All were negative for p40 except one case (focal weak positivity). PTEN was markedly reduced in background secretory glands in all cases; in 5/7, LEPP foci showed complete absence of PTEN expression. PIK3CA pathogenic variants were identified in 4/4 cases sequenced; 3/4 had inactivating PTEN mutations. Follow-up, available in 8 patients (mean length = 51 months, range 7-161), was conservative with observation only, and showed no persistence or adverse outcomes. LEPP is characterized by intraglandular cribriform/solid architecture, positive ER/PR, PTEN loss, and PIK3CA &PTEN mutations. While our findings indicate that LEPP is of neoplastic nature, for now we advise against diagnosing LEPP as endometrial carcinoma or hyperplasia since LEPP has a particular clinico-pathologic context (concurrent gestation), distinct morphology (purely intraepithelial complex growth) and indolent outcome. Thus, it should be distinguished from endometrial intraepithelial neoplasia and carcinoma for which therapeutic interventions are indicated.
    Keywords:  Endometrial proliferation of pregnancy; PIK3CA; PTEN; endometrial intraepithelial neoplasia; progesterone
  10. Trends Cell Biol. 2023 May 13. pii: S0962-8924(23)00080-6. [Epub ahead of print]
      Evolutionary cell biology explores the origins, principles, and core functions of cellular features and regulatory networks through the lens of evolution. This emerging field relies heavily on comparative experiments and genomic analyses that focus exclusively on extant diversity and historical events, providing limited opportunities for experimental validation. In this opinion article, we explore the potential for experimental laboratory evolution to augment the evolutionary cell biology toolbox, drawing inspiration from recent studies that combine laboratory evolution with cell biological assays. Primarily focusing on approaches for single cells, we provide a generalizable template for adapting experimental evolution protocols to provide fresh insight into long-standing questions in cell biology.
    Keywords:  adaptation; evolutionary cell biology; evolutionary dynamics; evolutionary innovation; experimental design
  11. Elife. 2023 May 18. pii: e82543. [Epub ahead of print]12
      Background: Sporadic venous malformation (VM) and angiomatosis of soft tissue (AST) are benign, congenital vascular anomalies affecting venous vasculature. Depending on the size and location of the lesion, symptoms vary from motility disturbances to pain and disfigurement. Due to high recurrence of the lesions more effective therapies are needed.Methods: As targeting stromal cells has been an emerging concept in anti-angiogenic therapies, here, by using VM/AST patient samples, RNA-sequencing, cell culture techniques and a xenograft mouse model, we investigated the crosstalk of endothelial cells (EC) and fibroblasts and its effect on vascular lesion growth.
    Results: We report, for the first time, expression and secretion of transforming growth factor A (TGFA) in ECs or intervascular stromal cells in AST and VM lesions. TGFA induced secretion of VEGF-A paracrinally, and regulated EC proliferation. Oncogenic PIK3CA variant in p.H1047R, a common somatic mutation found in these lesions, increased TGFA expression, enrichment of hallmark hypoxia, and in a mouse xenograft model, lesion size and vascularization. Treatment with afatinib, a pan-ErbB tyrosine-kinase inhibitor, decreased vascularization and lesion size in mouse xenograft model with ECs expressing oncogenic PIK3CA p.H1047R variant and fibroblasts.
    Conclusions: Based on the data, we suggest that targeting of both intervascular stromal cells and ECs is a potential treatment strategy for vascular lesions having a fibrous component.
    Funding: Academy of Finland, Ella and Georg Ehnrooth foundation, the ERC grants, Sigrid Jusélius Foundation, Finnish Foundation for Cardiovascular Research, Jane and Aatos Erkko Foundation, and Department of Musculosceletal and Plastic Surgery, Helsinki University Hospital.
    Keywords:  cell biology; human; medicine; mouse
  12. bioRxiv. 2023 May 03. pii: 2023.05.03.538868. [Epub ahead of print]
      Background: Lymphangiogenesis is believed to be a protective response in the setting of multiple forms of kidney injury and mitigates the progression of interstitial fibrosis. To augment this protective response, promoting kidney lymphangiogenesis is being investigated as a potential treatment to slow the progression of kidney disease.As injury related lymphangiogenesis is driven by signaling from the receptor VEGFR-3 in response to the cognate growth factor VEGF-C released by tubular epithelial cells, this signaling pathway is a candidate for future kidney therapeutics. However, the consequences to kidney development and function to targeting this signaling pathway remains poorly defined.Methods: We generated a new mouse model expressing Vegf-C under regulation of the nephron progenitor Six2Cre driver strain ( Six2Vegf-C ). Mice underwent a detailed phenotypic evaluation. Whole kidneys were processed for histology and micro computed tomography 3-dimensional imaging.
    Results: Six2Vegf-C mice had reduced body weight and kidney function compared to littermate controls. Six2Vegf-C kidneys demonstrated large peripelvic fluid filled lesions with distortion of the pelvicalcyceal system which progressed in severity with age. 3D imaging showed a 3-fold increase in total cortical vascular density. Histology confirmed a substantial increase in LYVE1+/PDPN+/VEGFR3+ lymphatic capillaries extending alongside EMCN+ peritubular capillaries. There was no change in EMCN+ peritubular capillary density.
    Conclusions: Kidney lymphangiogenesis was robustly induced in the Six2Vegf-C mice. There were no changes in peritubular blood capillary density despite these endothelial cells also expressing VEGFR-3. The model resulted in a severe cystic kidney phenotype that resembled a human condition termed renal lymphangiectasia. This study defines the vascular consequences of augmenting VEGF-C signaling during kidney development and provides new insight into a mimicker of human cystic kidney disease.
  13. Nature. 2023 May 17.
      Focal copy-number amplification is an oncogenic event. Although recent studies have revealed the complex structure1-3 and the evolutionary trajectories4 of oncogene amplicons, their origin remains poorly understood. Here we show that focal amplifications in breast cancer frequently derive from a mechanism-which we term translocation-bridge amplification-involving inter-chromosomal translocations that lead to dicentric chromosome bridge formation and breakage. In 780 breast cancer genomes, we observe that focal amplifications are frequently connected to each other by inter-chromosomal translocations at their boundaries. Subsequent analysis indicates the following model: the oncogene neighbourhood is translocated in G1 creating a dicentric chromosome, the dicentric chromosome is replicated, and as dicentric sister chromosomes segregate during mitosis, a chromosome bridge is formed and then broken, with fragments often being circularized in extrachromosomal DNAs. This model explains the amplifications of key oncogenes, including ERBB2 and CCND1. Recurrent amplification boundaries and rearrangement hotspots correlate with oestrogen receptor binding in breast cancer cells. Experimentally, oestrogen treatment induces DNA double-strand breaks in the oestrogen receptor target regions that are repaired by translocations, suggesting a role of oestrogen in generating the initial translocations. A pan-cancer analysis reveals tissue-specific biases in mechanisms initiating focal amplifications, with the breakage-fusion-bridge cycle prevalent in some and the translocation-bridge amplification in others, probably owing to the different timing of DNA break repair. Our results identify a common mode of oncogene amplification and propose oestrogen as its mechanistic origin in breast cancer.
  14. bioRxiv. 2023 May 03. pii: 2023.05.03.539283. [Epub ahead of print]
      Pluripotent stem cells are defined by both the ability to unlimitedly self-renew and differentiate to any somatic cell lineage, but understanding the mechanisms that control stem cell fitness versus the pluripotent cell identity is challenging. We performed four parallel genome-scale CRISPR-Cas9 screens to investigate the interplay between these two aspects of pluripotency. Our comparative analyses led to the discovery of genes with distinct roles in pluripotency regulation, including many mitochondrial and metabolism regulators crucial for stem cell fitness, and chromatin regulators that control stem cell identity. We further discovered a core set of factors that control both stem cell fitness and pluripotency identity, including an interconnected network of chromatin factors that safeguard pluripotency. Our unbiased and systematic screening and comparative analyses disentangle two interconnected aspects of pluripotency, provide rich datasets for exploring pluripotent cell identity versus self-renewal, and offer a valuable model for categorizing gene function in broad biological contexts.
  15. Cell Rep. 2023 May 12. pii: S2211-1247(23)00520-X. [Epub ahead of print]42(5): 112509
      In tissue development and homeostasis, transforming growth factor (TGF)-β signaling is finely coordinated by latent forms and matrix sequestration. Optogenetics can offer precise and dynamic control of cell signaling. We report the development of an optogenetic human induced pluripotent stem cell system for TGF-β signaling and demonstrate its utility in directing differentiation into the smooth muscle, tenogenic, and chondrogenic lineages. Light-activated TGF-β signaling resulted in expression of differentiation markers at levels close to those in soluble factor-treated cultures, with minimal phototoxicity. In a cartilage-bone model, light-patterned TGF-β gradients allowed the establishment of hyaline-like layer of cartilage tissue at the articular surface while attenuating with depth to enable hypertrophic induction at the osteochondral interface. By selectively activating TGF-β signaling in co-cultures of light-responsive and non-responsive cells, undifferentiated and differentiated cells were simultaneously maintained in a single culture with shared medium. This platform can enable patient-specific and spatiotemporally precise studies of cellular decision making.
    Keywords:  CP: Developmental biology; TGF-β signaling; differentiation; human induced pluripotent stem cells; mesenchymal stem cells; optogenetics