bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2022–01–09
fifteen papers selected by
Ralitsa Radostinova Madsen, University College London
  1. PIP3 abundance overcomes PI3K signaling selectivity in invadopodia.
  2. Glucose starvation induces a switch in the histone acetylome for activation of gluconeogenic and fat metabolism genes.
  3. scDALI: modeling allelic heterogeneity in single cells reveals context-specific genetic regulation.
  4. DNA-PK promotes activation of the survival kinase AKT in response to DNA damage through an mTORC2-ECT2 pathway.
  5. Altering phosphoinositides in high-fat diet-associated prostate tumor xenograft growth.
  6. Metabolic diversity within breast cancer brain-tropic cells determines metastatic fitness.
  7. Identification of a Cowden syndrome patient with a novel PTEN mutation and establishment of patient-derived induced pluripotent stem cells.
  8. Disruption of the IL-33-ST2-AKT signaling axis impairs neurodevelopment by inhibiting microglial metabolic adaptation and phagocytic function.
  9. β-catenin links cell seeding density to global gene expression during mouse embryonic stem cell differentiation.
  10. Transcriptional census of epithelial-mesenchymal plasticity in cancer.
  11. Independent Drug Action in Combination Therapy: Implications for Precision Oncology.
  12. Evaluation of CRISPR gene-editing tools in zebrafish.
  13. Research-Relevant Conditions and Pathology of Laboratory Mice, Rats, Gerbils, Guinea Pigs, Hamsters, Naked Mole Rats, and Rabbits.
  14. Automated Assessment of Cancer Drug Efficacy On Breast Tumor Spheroids in Aggrewell™400 Plates Using Image Cytometry.
  15. The PIKK-AKT connection in the DNA damage response.
  1. FEBS Lett. 2022 Jan 06.
    PIP3 abundance overcomes PI3K signaling selectivity in invadopodia.
    Charles T Jakubik, Claire C Weckerly, Gerald R V Hammond, Anne R Bresnick, Jonathan M Backer.
      PI3Kβ is required for invadopodia-mediated matrix degradation by breast cancer cells. Invadopodia maturation requires GPCR activation of PI3Kβ and its coupling to SHIP2 to produce PI(3,4)P2 . We now test whether selectivity for PI3Kβ is preserved under conditions of mutational increases in PI3K activity. In breast cancer cells where PI3Kβ is inhibited, short chain diC8-PIP3  rescues gelatin degradation in a SHIP2-dependent manner; rescue by diC8-PI(3,4)P2  is SHIP2-independent. Surprisingly, expression of either activated PI3Kβ or PI3Kα mutants rescued the effects of PI3Kβ inhibition. In both cases, gelatin degradation was SHIP2-dependent. These data confirm the requirement for PIP3 conversion to PI(3,4)P2 for invadopodia function, and suggest that selectivity for distinct PI3K isotypes may be obviated by mutational activation of the PI3K pathway.
    Keywords:  PI 3-kinase; invadopodia; matrix degradation
    DOI:  https://doi.org/10.1002/1873-3468.14273
  2. Mol Cell. 2022 Jan 06. pii: S1097-2765(21)01077-7. [Epub ahead of print]82(1): 60-74.e5
    Glucose starvation induces a switch in the histone acetylome for activation of gluconeogenic and fat metabolism genes.
    Wen-Chuan Hsieh, Benjamin M Sutter, Holly Ruess, Spencer D Barnes, Venkat S Malladi, Benjamin P Tu.
      Acetyl-CoA is a key intermediate situated at the intersection of many metabolic pathways. The reliance of histone acetylation on acetyl-CoA enables the coordination of gene expression with metabolic state. Abundant acetyl-CoA has been linked to the activation of genes involved in cell growth or tumorigenesis through histone acetylation. However, the role of histone acetylation in transcription under low levels of acetyl-CoA remains poorly understood. Here, we use a yeast starvation model to observe the dramatic alteration in the global occupancy of histone acetylation following carbon starvation; the location of histone acetylation marks shifts from growth-promoting genes to gluconeogenic and fat metabolism genes. This reallocation is mediated by both the histone deacetylase Rpd3p and the acetyltransferase Gcn5p, a component of the SAGA transcriptional coactivator. Our findings reveal an unexpected switch in the specificity of histone acetylation to promote pathways that generate acetyl-CoA for oxidation when acetyl-CoA is limiting.
    Keywords:  Gcn5p; Rpd3p; SAGA; acetyl-CoA; environmental stress response; fat metabolism; gluconeogenesis; glucose starvation; histone acetylation; transcription
    DOI:  https://doi.org/10.1016/j.molcel.2021.12.015
  3. Genome Biol. 2022 Jan 06. 23(1): 8
    scDALI: modeling allelic heterogeneity in single cells reveals context-specific genetic regulation.
    Tobias Heinen, Stefano Secchia, James P Reddington, Bingqing Zhao, Eileen E M Furlong, Oliver Stegle.
      While it is established that the functional impact of genetic variation can vary across cell types and states, capturing this diversity remains challenging. Current studies using bulk sequencing either ignore this heterogeneity or use sorted cell populations, reducing discovery and explanatory power. Here, we develop scDALI, a versatile computational framework that integrates information on cellular states with allelic quantifications of single-cell sequencing data to characterize cell-state-specific genetic effects. We apply scDALI to scATAC-seq profiles from developing F1 Drosophila embryos and scRNA-seq from differentiating human iPSCs, uncovering heterogeneous genetic effects in specific lineages, developmental stages, or cell types.
    Keywords:  Single-cell, Regulatory genomics, Statistical methods
    DOI:  https://doi.org/10.1186/s13059-021-02593-8
  4. Sci Signal. 2022 Jan 04. 15(715): eabh2290
    DNA-PK promotes activation of the survival kinase AKT in response to DNA damage through an mTORC2-ECT2 pathway.
    Liu Liu, Xiaoming Dai, Shasha Yin, Pengda Liu, Elizabeth G Hill, Wenyi Wei, Wenjian Gan.
      [Figure: see text].
    DOI:  https://doi.org/10.1126/scisignal.abh2290
  5. MedComm (2020). 2021 Dec;2(4): 756-764
    Altering phosphoinositides in high-fat diet-associated prostate tumor xenograft growth.
    Mingguo Huang, Atsushi Koizumi, Shintaro Narita, Hiroki Nakanishi, Hiromi Sato, Soki Kashima, Taketoshi Nara, Sohei Kanda, Kazuyuki Numakura, Mitsuru Saito, Shigeru Satoh, Hiroshi Nanjo, Takehiko Sasaki, Tomonori Habuchi.
      The metabolic reprogramming of phospholipids may affect intracellular signal transduction pathways. A high-fat diet (HFD) is attributed to prostate cancer (PCa) progression, but the expression pattern and role of phospholipids in HFD-mediated PCa progression remains unclear. In this study, HFD enhanced LNCaP xenograft tumor growth by upregulating the phosphatidylinositol (PI) 3-kinase (PI3K)/AKT signaling pathway. A lipidomic analysis using xenograft tumors showed that phosphoinositides, especially PI (3,4,5)-trisphosphate (PIP3), including several species containing C38:4, C38:3, and C40:4 fatty acids, increased in the HFD group compared to control. Fatty acid synthase (FASN) was significantly upregulated in xenograft tumors under HFD in both gene and protein levels. PCa cell growth was significantly inhibited through the decreased AKT signaling pathway by treatment with cerulenin, a chemical FASN inhibitor, which also downregulated PIP, PIP2, and PIP3 but not PI. Thus, dietary fat influences PCa progression and alters phosphoinositides, especially PIP3, a critical player in the PI3K/AKT pathway. These results may offer appropriate targets, such as FASN, for dietary intervention and/or chemoprevention to reduce PCa incidence and progression.
    Keywords:  AKT; FASN; high‐fat diet; phosphoinositide; prostate cancer
    DOI:  https://doi.org/10.1002/mco2.89
  6. Cell Metab. 2022 Jan 04. pii: S1550-4131(21)00620-3. [Epub ahead of print]34(1): 90-105.e7
    Metabolic diversity within breast cancer brain-tropic cells determines metastatic fitness.
    Pravat Kumar Parida, Mauricio Marquez-Palencia, Vidhya Nair, Akash K Kaushik, Kangsan Kim, Jessica Sudderth, Eduardo Quesada-Diaz, Ambar Cajigas, Vamsidhara Vemireddy, Paula I Gonzalez-Ericsson, Melinda E Sanders, Bret C Mobley, Kenneth Huffman, Sunati Sahoo, Prasanna Alluri, Cheryl Lewis, Yan Peng, Robert M Bachoo, Carlos L Arteaga, Ariella B Hanker, Ralph J DeBerardinis, Srinivas Malladi.
      HER2+ breast cancer patients are presented with either synchronous (S-BM), latent (Lat), or metachronous (M-BM) brain metastases. However, the basis for disparate metastatic fitness among disseminated tumor cells of similar oncotype within a distal organ remains unknown. Here, employing brain metastatic models, we show that metabolic diversity and plasticity within brain-tropic cells determine metastatic fitness. Lactate secreted by aggressive metastatic cells or lactate supplementation to mice bearing Lat cells limits innate immunosurveillance and triggers overt metastasis. Attenuating lactate metabolism in S-BM impedes metastasis, while M-BM adapt and survive as residual disease. In contrast to S-BM, Lat and M-BM survive in equilibrium with innate immunosurveillance, oxidize glutamine, and maintain cellular redox homeostasis through the anionic amino acid transporter xCT. Moreover, xCT expression is significantly higher in matched M-BM brain metastatic samples compared to primary tumors from HER2+ breast cancer patients. Inhibiting xCT function attenuates residual disease and recurrence in these preclinical models.
    Keywords:  HER2; breast cancer brain metastasis; immune surveillance; late recurrences; metabolism; metastasis; metastatic dormancy; metastatic latency; redox homeostasis; relapse
    DOI:  https://doi.org/10.1016/j.cmet.2021.12.001
  7. In Vitro Cell Dev Biol Anim. 2022 Jan 03.
    Identification of a Cowden syndrome patient with a novel PTEN mutation and establishment of patient-derived induced pluripotent stem cells.
    Fumitaka Obayashi, Atsuko Hamada, Sachiko Yamasaki, Taku Kanda, Shigeaki Toratani, Tetsuji Okamoto.
      Cowden syndrome (CS) is an autosomal dominant inherited disorder characterized by multiple hamartomas in various organs such as the mucosa, skin, and gastrointestinal tract. Patients with CS are at high risk for breast and thyroid cancers. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a tumor suppressor gene that negatively regulates the AKT pathway, and PTEN mutations are known to be the major causes of this syndrome. However, the pathogenesis of this syndrome has not been clarified. Here, we present a case of a Japanese woman with multiple oral polyps, breast cancer, and thyroid cancer who was clinically diagnosed with CS. We obtained DNA and RNA samples from the patient's peripheral blood mononuclear cells (PBMCs) and buccal mucosa tumor. Next-generation sequencing revealed novel germline mutations (c.1020delT and c.1026G > A) in exon 8 of PTEN. Sanger sequencing identified no PTEN transcript from the mutant allele. Furthermore, CS-specific induced pluripotent stem cells (CS-iPSCs) were established from PBMCs of the patient under feeder- and serum-free culture. Compared with healthy PBMCs and iPSCs, both of the CS-derived PBMCs and CS-iPSCs exhibited significantly reduced expression of the PTEN transcript. The transcriptional variant, PTENδ, was increased in CS-iPSCs, suggesting that it may be the cause of the disease.
    Keywords:  Cowden syndrome; Feeder- and serum-free culture condition; Induced pluripotent stem cells; Phosphatase and tensin homolog deleted on chromosome 10; Phosphatase and tensin homolog deleted on chromosome 10 δ
    DOI:  https://doi.org/10.1007/s11626-021-00637-8
  8. Immunity. 2021 Dec 24. pii: S1074-7613(21)00534-3. [Epub ahead of print]
    Disruption of the IL-33-ST2-AKT signaling axis impairs neurodevelopment by inhibiting microglial metabolic adaptation and phagocytic function.
    Danyang He, Heping Xu, Huiyuan Zhang, Ruihan Tang, Yangning Lan, Ruxiao Xing, Shaomin Li, Elena Christian, Yu Hou, Paul Lorello, Barbara Caldarone, Jiarui Ding, Lan Nguyen, Danielle Dionne, Pratiksha Thakore, Alexandra Schnell, Jun R Huh, Orit Rozenblatt-Rosen, Aviv Regev, Vijay K Kuchroo.
      To accommodate the changing needs of the developing brain, microglia must undergo substantial morphological, phenotypic, and functional reprogramming. Here, we examined whether cellular metabolism regulates microglial function during neurodevelopment. Microglial mitochondria bioenergetics correlated with and were functionally coupled to phagocytic activity in the developing brain. Transcriptional profiling of microglia with diverse metabolic profiles revealed an activation signature wherein the interleukin (IL)-33 signaling axis is associated with phagocytic activity. Genetic perturbation of IL-33 or its receptor ST2 led to microglial dystrophy, impaired synaptic function, and behavioral abnormalities. Conditional deletion of Il33 from astrocytes or Il1rl1, encoding ST2, in microglia increased susceptibility to seizures. Mechanistically, IL-33 promoted mitochondrial activity and phagocytosis in an AKT-dependent manner. Mitochondrial metabolism and AKT activity were temporally regulated in vivo. Thus, a microglia-astrocyte circuit mediated by the IL-33-ST2-AKT signaling axis supports microglial metabolic adaptation and phagocytic function during early development, with implications for neurodevelopmental and neuropsychiatric disorders.
    Keywords:  IL-33; bioenergenetics; microglia; neurodevelopment; phagocytosis; seizure; synapse
    DOI:  https://doi.org/10.1016/j.immuni.2021.12.001
  9. iScience. 2022 Jan 21. 25(1): 103541
    β-catenin links cell seeding density to global gene expression during mouse embryonic stem cell differentiation.
    Lucy LeBlanc, Mijeong Kim, Aparna Kambhampati, Albert J Son, Nereida Ramirez, Jonghwan Kim.
      Although cell density is known to affect numerous biological processes including gene expression and cell fate specification, mechanistic understanding of what factors link cell density to global gene regulation is lacking. Here, we reveal that the expression of thousands of genes in mouse embryonic stem cells (mESCs) is affected by cell seeding density and that low cell density enhances the efficiency of differentiation. Mechanistically, β-catenin is localized primarily to adherens junctions during both self-renewal and differentiation at high density. However, when mESCs differentiate at low density, β-catenin translocates to the nucleus and associates with Tcf7l1, inducing co-occupied lineage markers. Meanwhile, Esrrb sustains the expression of pluripotency-associated genes while repressing lineage markers at high density, and its association with DNA decreases at low density. Our results provide new insights into the previously neglected but pervasive phenomenon of density-dependent gene regulation.
    Keywords:  Cell biology; Stem cells research; Transcriptomics
    DOI:  https://doi.org/10.1016/j.isci.2021.103541
  10. Sci Adv. 2022 Jan 07. 8(1): eabi7640
    Transcriptional census of epithelial-mesenchymal plasticity in cancer.
    David P Cook, Barbara C Vanderhyden.
      [Figure: see text].
    DOI:  https://doi.org/10.1126/sciadv.abi7640
  11. Cancer Discov. 2022 Jan 04. pii: candisc.0212.2021. [Epub ahead of print]
    Independent Drug Action in Combination Therapy: Implications for Precision Oncology.
    Deborah Plana, Adam C Palmer, Peter K Sorger.
      Combination therapies are superior to monotherapy for many cancers. This advantage was historically ascribed to the ability of combinations to address tumor heterogeneity, but synergistic interaction is now a common explanation as well as a design criterion for new combinations. We review evidence that independent drug action, described in 1961, explains the efficacy of many practice-changing combination therapies: it provides populations of patients with heterogeneous drug sensitivities multiple chances of benefit from at least one drug. Understanding response heterogeneity could reveal predictive or pharmacodynamic biomarkers for more precise use of existing drugs and realize the benefits of additivity or synergy.
    DOI:  https://doi.org/10.1158/2159-8290.CD-21-0212
  12. BMC Genomics. 2022 Jan 06. 23(1): 12
    Evaluation of CRISPR gene-editing tools in zebrafish.
    José M Uribe-Salazar, Gulhan Kaya, Aadithya Sekar, KaeChandra Weyenberg, Cole Ingamells, Megan Y Dennis.
       BACKGROUND: Zebrafish have practical features that make them a useful model for higher-throughput tests of gene function using CRISPR/Cas9 editing to create 'knockout' models. In particular, the use of G0 mosaic mutants has potential to increase throughput of functional studies significantly but may suffer from transient effects of introducing Cas9 via microinjection. Further, a large number of computational and empirical tools exist to design CRISPR assays but often produce varied predictions across methods leaving uncertainty in choosing an optimal approach for zebrafish studies.
    METHODS: To systematically assess accuracy of tool predictions of on- and off-target gene editing, we subjected zebrafish embryos to CRISPR/Cas9 with 50 different guide RNAs (gRNAs) targeting 14 genes. We also investigate potential confounders of G0-based CRISPR screens by assaying control embryos for spurious mutations and altered gene expression.
    RESULTS: We compared our experimental in vivo editing efficiencies in mosaic G0 embryos with those predicted by eight commonly used gRNA design tools and found large discrepancies between methods. Assessing off-target mutations (predicted in silico and in vitro) found that the majority of tested loci had low in vivo frequencies (< 1%). To characterize if commonly used 'mock' CRISPR controls (larvae injected with Cas9 enzyme or mRNA with no gRNA) exhibited spurious molecular features that might exacerbate studies of G0 mosaic CRISPR knockout fish, we generated an RNA-seq dataset of various control larvae at 5 days post fertilization. While we found no evidence of spontaneous somatic mutations of injected larvae, we did identify several hundred differentially-expressed genes with high variability between injection types. Network analyses of shared differentially-expressed genes in the 'mock' injected larvae implicated a number of key regulators of common metabolic pathways, and gene-ontology analysis revealed connections with response to wounding and cytoskeleton organization, highlighting a potentially lasting effect from the microinjection process that requires further investigation.
    CONCLUSION: Overall, our results provide a valuable resource for the zebrafish community for the design and execution of CRISPR/Cas9 experiments.
    Keywords:  CIRCLE-seq; CRISPR; Cas9; Danio rerio; Gene knockout; RNA-seq; Zebrafish
    DOI:  https://doi.org/10.1186/s12864-021-08238-1
  13. ILAR J. 2022 Jan 04. pii: ilab022. [Epub ahead of print]
    Research-Relevant Conditions and Pathology of Laboratory Mice, Rats, Gerbils, Guinea Pigs, Hamsters, Naked Mole Rats, and Rabbits.
    Timothy K Cooper, David K Meyerholz, Amanda P Beck, Martha A Delaney, Alessandra Piersigilli, Teresa L Southard, Cory F Brayton.
      Animals are valuable resources in biomedical research in investigations of biological processes, disease pathogenesis, therapeutic interventions, safety, toxicity, and carcinogenicity. Interpretation of data from animals requires knowledge not only of the processes or diseases (pathophysiology) under study but also recognition of spontaneous conditions and background lesions (pathology) that can influence or confound the study results. Species, strain/stock, sex, age, anatomy, physiology, spontaneous diseases (noninfectious and infectious), and neoplasia impact experimental results and interpretation as well as animal welfare. This review and the references selected aim to provide a pathology resource for researchers, pathologists, and veterinary personnel who strive to achieve research rigor and validity and must understand the spectrum of "normal" and expected conditions to accurately identify research-relevant experimental phenotypes as well as unusual illness, pathology, or other conditions that can compromise studies involving laboratory mice, rats, gerbils, guinea pigs, hamsters, naked mole rats, and rabbits.
    Keywords:  Cricetinae; Gerbillinae; guinea pigs; mice; mole rats; rabbits; rats
    DOI:  https://doi.org/10.1093/ilar/ilab022
  14. J Fluoresc. 2022 Jan 06.
    Automated Assessment of Cancer Drug Efficacy On Breast Tumor Spheroids in Aggrewell™400 Plates Using Image Cytometry.
    Shilpaa Mukundan, Jordan Bell, Matthew Teryek, Charles Hernandez, Andrea C Love, Biju Parekkadan, Leo Li-Ying Chan.
      Tumor spheroid models have proven useful in the study of cancer cell responses to chemotherapeutic compounds by more closely mimicking the 3-dimensional nature of tumors in situ. Their advantages are often offset, however, by protocols that are long, complicated, and expensive. Efforts continue for the development of high-throughput assays that combine the advantages of 3D models with the convenience and simplicity of traditional 2D monolayer methods. Herein, we describe the development of a breast cancer spheroid image cytometry assay using T47D cells in Aggrewell™400 spheroid plates. Using the Celigo® automated imaging system, we developed a method to image and individually track thousands of spheroids within the Aggrewell™400 microwell plate over time. We demonstrate the use of calcein AM and propidium iodide staining to study the effects of known anti-cancer drugs Doxorubicin, Everolimus, Gemcitabine, Metformin, Paclitaxel and Tamoxifen. We use the image cytometry results to quantify the fluorescence of calcein AM and PI as well as spheroid size in a dose dependent manner for each of the drugs. We observe a dose-dependent reduction in spheroid size and find that it correlates well with the viability obtained from the CellTiter96® endpoint assay. The image cytometry method we demonstrate is a convenient and high-throughput drug-response assay for breast cancer spheroids under 400 μm in diameter, and may lay a foundation for investigating other three-dimensional spheroids, organoids, and tissue samples.
    Keywords:  Aggrewell™400; Celigo; High-throughput screening; Image cytometry; Tumor spheroid
    DOI:  https://doi.org/10.1007/s10895-021-02881-3
  15. Sci Signal. 2022 Jan 04. 15(715): eabm6211
    The PIKK-AKT connection in the DNA damage response.
    Sejeong Shin, Katherine A Walker, Sang-Oh Yoon.
      [Figure: see text].
    DOI:  https://doi.org/10.1126/scisignal.abm6211
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