bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2024–03–31
fifteen papers selected by
Ralitsa Radostinova Madsen, MRC-PPU
  1. Non-catalytic role of phosphoinositide 3-kinase in mesenchymal cell migration through non-canonical induction of p85β/AP2-mediated endocytosis.
  2. Optimal targeting of PI3K-AKT and mTOR in advanced oestrogen receptor-positive breast cancer.
  3. Activation of the insulin receptor by insulin-like growth factor 2.
  4. Improving CRISPR-Cas9 directed faithful transgene integration outcomes by reducing unwanted random DNA integration.
  5. DELVE: feature selection for preserving biological trajectories in single-cell data.
  6. Different oncogenes and reproductive histories shape the progression of distinct premalignant clones in multistage mouse breast cancer models.
  7. Pooled CRISPR screening of high-content cellular phenotypes using ghost cytometry.
  8. FixNCut: single-cell genomics through reversible tissue fixation and dissociation.
  9. PathIntegrate: Multivariate modelling approaches for pathway-based multi-omics data integration.
  10. Data integration strategies for whole-cell modeling.
  11. Gene-expression memory-based prediction of cell lineages from scRNA-seq datasets.
  12. Establishing conditions for the generation and maintenance of estrogen receptor-positive organoid models of breast cancer.
  13. More than just a simple barrier.
  14. Developmental and behavioral phenotypes of pediatric patients with PTEN hamartoma tumor syndrome.
  15. Paradoxical activation of oncogenic signaling as a cancer treatment strategy.
  1. Nat Commun. 2024 Mar 23. 15(1): 2612
    Non-catalytic role of phosphoinositide 3-kinase in mesenchymal cell migration through non-canonical induction of p85β/AP2-mediated endocytosis.
    Hideaki T Matsubayashi, Jack Mountain, Nozomi Takahashi, Abhijit Deb Roy, Tony Yao, Amy F Peterson, Cristian Saez Gonzalez, Ibuki Kawamata, Takanari Inoue.
      Class IA phosphoinositide 3-kinase (PI3K) galvanizes fundamental cellular processes such as migration, proliferation, and differentiation. To enable these multifaceted roles, the catalytic subunit p110 utilizes the multi-domain, regulatory subunit p85 through its inter SH2 domain (iSH2). In cell migration, its product PI(3,4,5)P3 generates locomotive activity. While non-catalytic roles are also implicated, underlying mechanisms and their relationship to PI(3,4,5)P3 signaling remain elusive. Here, we report that a disordered region of iSH2 contains AP2 binding motifs which can trigger clathrin and dynamin-mediated endocytosis independent of PI3K catalytic activity. The AP2 binding motif mutants of p85 aberrantly accumulate at focal adhesions and increase both velocity and persistency in fibroblast migration. We thus propose the dual functionality of PI3K in the control of cell motility, catalytic and non-catalytic, arising distinctly from juxtaposed regions within iSH2.
    DOI:  https://doi.org/10.1038/s41467-024-46855-y
  2. Lancet Oncol. 2024 Apr;pii: S1470-2045(23)00676-9. [Epub ahead of print]25(4): e139-e151
    Optimal targeting of PI3K-AKT and mTOR in advanced oestrogen receptor-positive breast cancer.
    Iseult M Browne, Fabrice André, Sarat Chandarlapaty, Lisa A Carey, Nicholas C Turner.
      The growing availability of targeted therapies for patients with advanced oestrogen receptor-positive breast cancer has improved survival, but there remains much to learn about the optimal management of these patients. The PI3K-AKT and mTOR pathways are among the most commonly activated pathways in breast cancer, whose crucial role in the pathogenesis of this tumour type has spurred major efforts to target this pathway at specific kinase hubs. Approvals for oestrogen receptor-positive advanced breast cancer include the PI3K inhibitor alpelisib for PIK3CA-mutated tumours, the AKT inhibitor capivasertib for tumours with alterations in PIK3CA, AKT1, or PTEN, and the mTOR inhibitor everolimus, which is used irrespective of mutation status. The availability of different inhibitors leaves physicians with a potentially challenging decision over which of these therapies should be used for individual patients and when. In this Review, we present a comprehensive summary of our current understanding of the pathways and the three inhibitors and discuss strategies for the optimal sequencing of therapies in the clinic, particularly after progression on a CDK4/6 inhibitor.
    DOI:  https://doi.org/10.1016/S1470-2045(23)00676-9
  3. Nat Commun. 2024 Mar 23. 15(1): 2609
    Activation of the insulin receptor by insulin-like growth factor 2.
    Weidong An, Catherine Hall, Jie Li, Albert Hung, Jiayi Wu, Junhee Park, Liwei Wang, Xiao-Chen Bai, Eunhee Choi.
      Insulin receptor (IR) controls growth and metabolism. Insulin-like growth factor 2 (IGF2) has different binding properties on two IR isoforms, mimicking insulin's function. However, the molecular mechanism underlying IGF2-induced IR activation remains unclear. Here, we present cryo-EM structures of full-length human long isoform IR (IR-B) in both the inactive and IGF2-bound active states, and short isoform IR (IR-A) in the IGF2-bound active state. Under saturated IGF2 concentrations, both the IR-A and IR-B adopt predominantly asymmetric conformations with two or three IGF2s bound at site-1 and site-2, which differs from that insulin saturated IR forms an exclusively T-shaped symmetric conformation. IGF2 exhibits a relatively weak binding to IR site-2 compared to insulin, making it less potent in promoting full IR activation. Cell-based experiments validated the functional importance of IGF2 binding to two distinct binding sites in optimal IR signaling and trafficking. In the inactive state, the C-terminus of α-CT of IR-B contacts FnIII-2 domain of the same protomer, hindering its threading into the C-loop of IGF2, thus reducing the association rate of IGF2 with IR-B. Collectively, our studies demonstrate the activation mechanism of IR by IGF2 and reveal the molecular basis underlying the different affinity of IGF2 to IR-A and IR-B.
    DOI:  https://doi.org/10.1038/s41467-024-46990-6
  4. J Biomed Sci. 2024 Mar 26. 31(1): 32
    Improving CRISPR-Cas9 directed faithful transgene integration outcomes by reducing unwanted random DNA integration.
    Rio Hermantara, Laura Richmond, Aqeel Faisal Taqi, Sabari Chilaka, Valentine Jeantet, Ileana Guerrini, Katherine West, Adam West.
       BACKGROUND: The field of genome editing has been revolutionized by the development of an easily programmable editing tool, the CRISPR-Cas9. Despite its promise, off-target activity of Cas9 posed a great disadvantage for genome editing purposes by causing DNA double strand breaks at off-target locations and causing unwanted editing outcomes. Furthermore, for gene integration applications, which introduce transgene sequences, integration of transgenes to off-target sites could be harmful, hard to detect, and reduce faithful genome editing efficiency.
    METHOD: Here we report the development of a multicolour fluorescence assay for studying CRISPR-Cas9-directed gene integration at an endogenous locus in human cell lines. We examine genetic integration of reporter genes in transiently transfected cells as well as puromycin-selected stable cell lines to determine the fidelity of multiple CRISPR-Cas9 strategies.
    RESULT: We found that there is a high occurrence of unwanted DNA integration which tarnished faithful knock-in efficiency. Integration outcomes are influenced by the type of DNA DSBs, donor design, the use of enhanced specificity Cas9 variants, with S-phase regulated Cas9 activity. Moreover, restricting Cas9 expression with a self-cleaving system greatly improves knock-in outcomes by substantially reducing the percentage of cells with unwanted DNA integration.
    CONCLUSION: Our results highlight the need for a more stringent assessment of CRISPR-Cas9-mediated knock-in outcomes, and the importance of careful strategy design to maximise efficient and faithful transgene integration.
    Keywords:  CRISPR–Cas9; Faithful genome editing; Homology arms; Knock-in; Off-target; Self-cleaving
    DOI:  https://doi.org/10.1186/s12929-024-01020-x
  5. Nat Commun. 2024 Mar 29. 15(1): 2765
    DELVE: feature selection for preserving biological trajectories in single-cell data.
    Jolene S Ranek, Wayne Stallaert, J Justin Milner, Margaret Redick, Samuel C Wolff, Adriana S Beltran, Natalie Stanley, Jeremy E Purvis.
      Single-cell technologies can measure the expression of thousands of molecular features in individual cells undergoing dynamic biological processes. While examining cells along a computationally-ordered pseudotime trajectory can reveal how changes in gene or protein expression impact cell fate, identifying such dynamic features is challenging due to the inherent noise in single-cell data. Here, we present DELVE, an unsupervised feature selection method for identifying a representative subset of molecular features which robustly recapitulate cellular trajectories. In contrast to previous work, DELVE uses a bottom-up approach to mitigate the effects of confounding sources of variation, and instead models cell states from dynamic gene or protein modules based on core regulatory complexes. Using simulations, single-cell RNA sequencing, and iterative immunofluorescence imaging data in the context of cell cycle and cellular differentiation, we demonstrate how DELVE selects features that better define cell-types and cell-type transitions. DELVE is available as an open-source python package: https://github.com/jranek/delve .
    DOI:  https://doi.org/10.1038/s41467-024-46773-z
  6. Am J Pathol. 2024 Mar 25. pii: S0002-9440(24)00112-3. [Epub ahead of print]
    Different oncogenes and reproductive histories shape the progression of distinct premalignant clones in multistage mouse breast cancer models.
    Maryknoll Linscott, Jerry Ruizhe Ren, Shelley Gestl, Edward J Gunther.
      A remote carcinogen exposure can predispose to breast cancer onset decades later, suggesting carcinogen-induced mutations generate long-lived premalignant clones. How subsequent events influence the progression of specific premalignant clones remains poorly understood. Here, we generated multistage mouse models of mammary carcinogenesis by combining chemical carcinogen exposure (using 7,12-dimethylbenzanthracene, DMBA) with transgenes that enable inducible expression of one of two clinically relevant mammary oncogenes: c-MYC (MYC) or PIK3CAH1047R (PIK). In prior work, DMBA exposure generated mammary clones bearing signature HrasQ61L mutations, which only progressed to mammary cancer after inducible Wnt1 oncogene expression. Here, after an identical DMBA exposure, MYC versus PIK drove cancer progression from mammary clones bearing mutations in distinct Ras family paralogs. For example, MYC drove cancer progression from either Kras- or Nras-mutant clones, whereas PIK transformed Kras-mutant clones only. These Ras mutation patterns were maintained whether oncogenic transgenes were induced within days of DMBA exposure or months later. Completing a full-term pregnancy (parity) failed to protect against either MYC- or PIK-driven tumor progression. Instead, a postpartum increase in mammary tumor predisposition was observed in the context of PIK-driven progression. However, parity decreased the overall prevalence of tumors bearing Krasmut, and the magnitude of this decrease depended on both the number and timing of pregnancies. These multistage models may be useful for elucidating biological features of premalignant mammary neoplasia.
    Keywords:  Cancer; Chemical carcinogenesis; Mammary; Mouse; Myc; Pik3ca; Ras
    DOI:  https://doi.org/10.1016/j.ajpath.2024.02.018
  7. Cell Rep Methods. 2024 Mar 25. pii: S2667-2375(24)00062-6. [Epub ahead of print]4(3): 100737
    Pooled CRISPR screening of high-content cellular phenotypes using ghost cytometry.
    Asako Tsubouchi, Yuri An, Yoko Kawamura, Yuichi Yanagihashi, Hirofumi Nakayama, Yuri Murata, Kazuki Teranishi, Soh Ishiguro, Hiroyuki Aburatani, Nozomu Yachie, Sadao Ota.
      Recent advancements in image-based pooled CRISPR screening have facilitated the mapping of diverse genotype-phenotype associations within mammalian cells. However, the rapid enrichment of cells based on morphological information continues to pose a challenge, constraining the capacity for large-scale gene perturbation screening across diverse high-content cellular phenotypes. In this study, we demonstrate the applicability of multimodal ghost cytometry-based cell sorting, including both fluorescent and label-free high-content phenotypes, for rapid pooled CRISPR screening within vast cell populations. Using the high-content cell sorter operating in fluorescence mode, we successfully executed kinase-specific CRISPR screening targeting genes influencing the nuclear translocation of RelA. Furthermore, using the multiparametric, label-free mode, we performed large-scale screening to identify genes involved in macrophage polarization. Notably, the label-free platform can enrich target phenotypes without requiring invasive staining, preserving untouched cells for downstream assays and expanding the potential for screening cellular phenotypes even when suitable markers are absent.
    Keywords:  CP: biotechnology; CP: imaging; cellular phenotyping; flow cytometry; high-content cell analysis; image-based cell sorter; label-free cell analysis; machine learning; pooled CRISPR screening
    DOI:  https://doi.org/10.1016/j.crmeth.2024.100737
  8. Genome Biol. 2024 Mar 29. 25(1): 81
    FixNCut: single-cell genomics through reversible tissue fixation and dissociation.
    Laura Jiménez-Gracia, Domenica Marchese, Juan C Nieto, Ginevra Caratù, Elisa Melón-Ardanaz, Victoria Gudiño, Sara Roth, Kellie Wise, Natalie K Ryan, Kirk B Jensen, Xavier Hernando-Momblona, Joana P Bernardes, Florian Tran, Laura Katharina Sievers, Stefan Schreiber, Maarten van den Berge, Tessa Kole, Petra L van der Velde, Martijn C Nawijn, Philip Rosenstiel, Eduard Batlle, Lisa M Butler, Ian A Parish, Jasmine Plummer, Ivo Gut, Azucena Salas, Holger Heyn, Luciano G Martelotto.
      The use of single-cell technologies for clinical applications requires disconnecting sampling from downstream processing steps. Early sample preservation can further increase robustness and reproducibility by avoiding artifacts introduced during specimen handling. We present FixNCut, a methodology for the reversible fixation of tissue followed by dissociation that overcomes current limitations. We applied FixNCut to human and mouse tissues to demonstrate the preservation of RNA integrity, sequencing library complexity, and cellular composition, while diminishing stress-related artifacts. Besides single-cell RNA sequencing, FixNCut is compatible with multiple single-cell and spatial technologies, making it a versatile tool for robust and flexible study designs.
    Keywords:  Cellular stress; RNA sequencing; Sample fixation; Single-cell genomics; Tissue dissociation
    DOI:  https://doi.org/10.1186/s13059-024-03219-5
  9. PLoS Comput Biol. 2024 Mar 25. 20(3): e1011814
    PathIntegrate: Multivariate modelling approaches for pathway-based multi-omics data integration.
    Cecilia Wieder, Juliette Cooke, Clement Frainay, Nathalie Poupin, Russell Bowler, Fabien Jourdan, Katerina J Kechris, Rachel Pj Lai, Timothy Ebbels.
      As terabytes of multi-omics data are being generated, there is an ever-increasing need for methods facilitating the integration and interpretation of such data. Current multi-omics integration methods typically output lists, clusters, or subnetworks of molecules related to an outcome. Even with expert domain knowledge, discerning the biological processes involved is a time-consuming activity. Here we propose PathIntegrate, a method for integrating multi-omics datasets based on pathways, designed to exploit knowledge of biological systems and thus provide interpretable models for such studies. PathIntegrate employs single-sample pathway analysis to transform multi-omics datasets from the molecular to the pathway-level, and applies a predictive single-view or multi-view model to integrate the data. Model outputs include multi-omics pathways ranked by their contribution to the outcome prediction, the contribution of each omics layer, and the importance of each molecule in a pathway. Using semi-synthetic data we demonstrate the benefit of grouping molecules into pathways to detect signals in low signal-to-noise scenarios, as well as the ability of PathIntegrate to precisely identify important pathways at low effect sizes. Finally, using COPD and COVID-19 data we showcase how PathIntegrate enables convenient integration and interpretation of complex high-dimensional multi-omics datasets. PathIntegrate is available as an open-source Python package.
    DOI:  https://doi.org/10.1371/journal.pcbi.1011814
  10. FEMS Yeast Res. 2024 Mar 27. pii: foae011. [Epub ahead of print]
    Data integration strategies for whole-cell modeling.
    Katja Tummler, Edda Klipp.
      Data makes the world go round-and high quality data is a prerequisite for precise models, especially for whole-cell models (WCM). Data for WCM must be reusable, contain information about the exact experimental background and should-in its entirety-cover all relevant processes in the cell. Here, we review basic requirements to data for WCM and strategies how to combine them. As a species-specific resource, we introduce the Yeast Cell Model Data Base (YCMDB) to illustrate requirements and solutions. We discuss recent standards for data as well as for computational models including the modeling process as data to be reported. We outline strategies for constructions of WCM despite their inherent complexity.
    Keywords:  complex networks; emerging standards; single cell; yeast cell model
    DOI:  https://doi.org/10.1093/femsyr/foae011
  11. Nat Commun. 2024 Mar 29. 15(1): 2744
    Gene-expression memory-based prediction of cell lineages from scRNA-seq datasets.
    A S Eisele, M Tarbier, A A Dormann, V Pelechano, D M Suter.
      Assigning single cell transcriptomes to cellular lineage trees by lineage tracing has transformed our understanding of differentiation during development, regeneration, and disease. However, lineage tracing is technically demanding, often restricted in time-resolution, and most scRNA-seq datasets are devoid of lineage information. Here we introduce Gene Expression Memory-based Lineage Inference (GEMLI), a computational tool allowing to robustly identify small to medium-sized cell lineages solely from scRNA-seq datasets. GEMLI allows to study heritable gene expression, to discriminate symmetric and asymmetric cell fate decisions and to reconstruct individual multicellular structures from pooled scRNA-seq datasets. In human breast cancer biopsies, GEMLI reveals previously unknown gene expression changes at the onset of cancer invasiveness. The universal applicability of GEMLI allows studying the role of small cell lineages in a wide range of physiological and pathological contexts, notably in vivo. GEMLI is available as an R package on GitHub ( https://github.com/UPSUTER/GEMLI ).
    DOI:  https://doi.org/10.1038/s41467-024-47158-y
  12. Breast Cancer Res. 2024 Mar 29. 26(1): 56
    Establishing conditions for the generation and maintenance of estrogen receptor-positive organoid models of breast cancer.
    Michael U J Oliphant, Dipikaa Akshinthala, Senthil K Muthuswamy.
      Patient-derived organoid models of estrogen receptor-positive (ER+) breast cancer would provide a much-needed tool to understand drug resistance and disease progression better. However, the establishment and long-term maintenance of ER expression, function, and response in vitro remains a significant challenge. Here, we report the development of an ER+ breast tumor organoid medium (BTOM-ER) that conserves ER expression, estrogen responsiveness, and dependence, as well as sensitivity to endocrine therapy of ER+ patient-derived xenograft organoids (PDXO). Our findings demonstrate the utility of subtype-specific culture conditions that better mimic the characteristics of the breast epithelial biology and microenvironment, providing a powerful platform for investigating therapy response and disease progression of ER+ breast cancer.
    DOI:  https://doi.org/10.1186/s13058-024-01798-6
  13. Elife. 2024 Mar 27. pii: e97247. [Epub ahead of print]13
    More than just a simple barrier.
    Erandi Velazquez-Miranda, Ming He.
      Endothelial cell subpopulations are characterized by unique gene expression profiles, epigenetic landscapes and functional properties.
    Keywords:  ERG; atherosclerosis; chromosomes; endothelial cells; epigenetics; gene expression; genetics; genomics; human; inflammation; vascular biology
    DOI:  https://doi.org/10.7554/eLife.97247
  14. Am J Med Genet A. 2024 Mar 28. e63608
    Developmental and behavioral phenotypes of pediatric patients with PTEN hamartoma tumor syndrome.
    Suzanne P MacFarland, Melani Duvall, Raissa Tchetcho Kemajou, Sarah E Baldino, Kristin Zelley, Chelsea Black, Allison Thomas, Nina H Thomas, Melanie Ruffner, Yimei Li, Judith S Miller, Garrett M Brodeur, Emily Shabason.
      Our study characterized the neurodevelopmental spectrum of individuals with PTEN Hamartoma Tumor Syndrome (PHTS), a syndrome that predisposes to both neurodevelopmental phenotypes and cancer risk. We aim to better understand life-impacting neurodevelopmental features of PHTS. Our study recruited 20 children/adolescents with PHTS, who were then administered assessments for autism spectrum disorder (ASD) and other neurocognitive measures, including assessment of IQ, executive and adaptive functioning, and health-related quality of life. Thirteen individuals (65%) were identified as having ASD, of which five were newly diagnosed during the study. Of those, ASD symptom severity was in the mild-moderate range for 77%. Overall, IQ was in the average range, with a mean of 92.61 (SD 24.45, p = 0.5), though there was a non-statistically significant trend toward individuals without ASD having a higher mean IQ (102.7 vs 82.3; p = 0.1). Subjects had significant impairment in processing speed (mean 75.38, SD 24.75, p < 0.05), decreased adaptive functioning skills across all domains, and a trend toward having more executive functioning problems. Individuals with PHTS are at increased risk of neurodevelopmental disorders, including ASD and impaired executive and adaptive functioning. Although clear guidelines exist for cancer surveillance for individuals with PHTS, additional guidelines and screening for neurodevelopmental disorders are warranted.
    Keywords:  PTEN hamartoma tumor syndrome; autism spectrum disorder
    DOI:  https://doi.org/10.1002/ajmg.a.63608
  15. Cancer Discov. 2024 Mar 27.
    Paradoxical activation of oncogenic signaling as a cancer treatment strategy.
    Matheus Henrique Dias, Anoek Friskes, Siying Wang, Joao M Fernandes Neto, Frank van Gemert, Soufiane Mourragui, Chrysa Papagianni, Hendrik J Kuiken, Sara Mainardi, Daniel Alvarez-Villanueva, Cor Lieftink, Ben Morris, Anna Dekker, Emma van Dijk, Lieke H S Wilms, Marcelo S da Silva, Robin A Jansen, Antonio Mulero-Sanchez, Elke Malzer, August Vidal, Cristina Santos, Ramon Salazar, Rosangela A M Wailemann, Thompson E P Torres, Giulia De Conti, Jonne A Raaijmakers, Petur Snaebjornsson, Shengxian Yuan, Wenxin Qin, John S Kovach, Hugo A Armelin, Hein Te Riele, Alexander van Oudernaarden, Haojie Jin, Roderick L Beijersbergen, Alberto Villanueva, Rene H Medema, Rene Bernards.
      Cancer homeostasis depends on a balance between activated oncogenic pathways driving tumorigenesis and engagement of stress-response programs that counteract the inherent toxicity of such aberrant signaling. While inhibition of oncogenic signaling pathways has been explored extensively, there is increasing evidence that overactivation of the same pathways can also disrupt cancer homeostasis and cause lethality. We show here that inhibition of Protein Phosphatase 2A (PP2A) hyperactivates multiple oncogenic pathways and engages stress responses in colon cancer cells. Genetic and compound screens identify combined inhibition of PP2A and WEE1 as synergistic in multiple cancer models by collapsing DNA replication and triggering premature mitosis followed by cell death. This combination also suppressed the growth of patient-derived tumors in vivo. Remarkably, acquired resistance to this drug combination suppressed the ability of colon cancer cells to form tumors in vivo. Our data suggest that paradoxical activation of oncogenic signaling can result in tumor suppressive resistance.
    DOI:  https://doi.org/10.1158/2159-8290.CD-23-0216
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